Selectable marker proteins, expression vectors, engineered cells and extracellular vesicles for the production of virus-like particles for therapeutic and prophylactic applications

ABSTRACT

The present invention relates to engineered selectable marker proteins for recombinant protein expression, as well as novel expression vector designs for achieving high-level recombinant protein expression, and cells transfected therewith as a platform technology for producing extracellular vesicle-based therapeutic or prophylactic compositions, wherein one or more recombinant proteins of interest are displayed on the surface of the extracellular vesicles. As an example, the present invention relates to a virus-like article composition comprising such extracellular vesicles displaying one or more antigens configured to induce immune responses against SARS-CoV-2.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority under 35 U.S.C. § 119(e)of U.S. Provisional Patent Application Ser. No. 63/155,268, filed Mar.1, 2021; U.S. Provisional Patent Application Ser. No. 63/110,325, filedNov. 5, 2020; U.S. Provisional Patent Application Ser. No. 63/108,847,filed Nov. 2, 2020; U.S. Provisional Patent Application Ser. No.63/061,766, filed Aug. 5, 2020; U.S. Provisional Patent Application Ser.No. 63/000,211, filed Mar. 26, 2020; U.S. Provisional Patent ApplicationSer. No. 62/990,946, filed Mar. 17, 2020; and U.S. Provisional PatentApplication Ser. No. 62/989,525, filed Mar. 13, 2020, the contents ofwhich are incorporated herein by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates generally to vaccines and morespecifically to non-naturally occurring, selectable marker (SM) proteinsand methods of use thereof.

BACKGROUND INFORMATION

The creation of transgenic mammalian cell lines was pioneered almostfour decades ago by Berg and colleagues (1-4). In general, this processinvolves transfecting (or transducing) cells with a recombinant DNAvector that carries the gene of interest and a selectable marker geneand then selecting for transgene-expressing cells using an appropriateantibiotic (3,4). This approach has been used thousands of times tocreate useful transgenic cell lines and is still in use today. However,most of the antibiotic-resistant clones arising from such experimentsexpress low or undetectable levels of the linked transgene, requiringthe isolation, expansion, and screening of dozens of independent,antibiotic-resistant, single-cell clones to find one that displays thedesired level of transgene expression (5).

Many researchers have devoted significant effort towards improving theoutcomes of mammalian cell transgenesis experiments. Much of this efforthas been directed at improved vector design, resulting in theidentification of many cis-acting features that affect transgeneexpression, including transcriptional regulatory regions (6-10), mRNApolyadenylation sites(11), introns (12), and mRNA export and/ortranslation signals (e.g. the Woodchuck Hepatitis Virus (WHP)posttranscriptional regulatory element (WPRE) (13)), and inhibitors ofgene silencing(10,14). Other studies have revealed that gene silencingcan be induced where bacterial and viral sequences in the transgenesisvectors can induce transgene silencing(15,16), effects that can beminimized by gene delivery via DNA mini-circles(17) or DNA transposons(e.g. Sleeping Beauty(18,19) or PiggyBac(20,21)). Furthermore, transgenedelivery via replicating episomes (e.g. plasmids carrying Epstein-Barrvirus (EBV) origin of replication (OriP) and the EBV nuclear antigen 1gene (EBNA1)) may boost transgene expression via elevated gene dosageeffects (22). Researchers have also invented technologies that directtransgene integration into sites of the host cell chromosome that arecompatible with high-level, stable transgene expression, such asrecombinase-mediated cassette exchange(23), phage ϕC31-mediated DNAintegration(24), and CRISPR/Cas9-mediated genome editing(25). Althoughthese site-directed insertional strategies represent a significanttechnological advance, several are limited to specific, previouslyengineered recipient cell lines, and all require the isolation,expansion, and characterization of numerous single cell clones.

Given the importance of mammalian cell transgenesis to biomedicalresearch, it is somewhat surprising that there is as yet no robust bodyof empirical studies comparing the relative effectiveness of thedominant selectable markers that are typically used in theseexperiments. Currently there are five dominant selectable markers inwidespread use, the NeoR, BsdR, HygR, PuroR, and BleoR genes, whichconfer resistance to the drugs G418, blasticidin, hygromycin B,puromycin, and zeocin, respectively (3,26-29). However, there is noclear understanding of whether they all work equally well for mammaliancell transgenesis experiments, or if they have differential effects ontransgene expression. As a result, the choice of selectable marker inmany mammalian cell transgenesis experiments tends to be based oncircumstance rather than evidence.

Consequently, there is a pressing need to develop new expression vectorswith improved selectable markers that could have a significant effect onthe expression of recombinant proteins in both cells and exosomes. Thepresent inventors sought to understand how the choice of selectablemarker and the process of antibiotic selection affects the expression oflinked recombinant proteins. The present inventors performed the studiesdescribed below in HEK293 cell lines because they are commonly used forbiochemical and cell biological studies and are an approved cell factoryfor producing biological materials & drugs (5,30). Furthermore, thepresent inventors performed many of these studies in the context of arecombinant exosomal cargo protein, CD81, as the present inventors havea longstanding interest in exosome biology and exosome engineering(31-44).

Exosomes are small, secreted vesicles of ˜30-150 nm in diameter that arereleased by all human cell types, contain discrete subsets of proteins,nucleic acids, and lipids, and can transmit signals and molecules toother cells in a pathway of intercellular vesicle traffic, and are ofincreasing use as potential therapeutics and drug delivery vehicles(42).

Vaccination is an effective way to provide prophylactic protectionagainst infectious diseases, including, but not limited to, viral,bacterial, and/or parasitic diseases, such as influenza, AIDS, hepatitisvirus infection, cholera, malaria, tuberculosis, and many otherdiseases. For example, influenza infections are the seventh leadingcause of death in the United States with 200,000 hospitalizations and40,000 deaths seen in the United States per year and cause about 3-5million hospitalizations and about 300,000 to 500,000 deaths worldwideper year. Millions of people receive flu vaccines to protect them fromseasonal flu each year. Vaccination also holds the best potential forpreventing the spread of coronavirus disease 2019 (COVID-19) caused bythe SARS coronavirus 2 (SARS-CoV-2).

A typical vaccine contains an agent that resembles the disease-causingagent, which could be a microorganism, such as bacteria, virus, fungi,parasites, or one or more toxins. The antigen or agent in the vaccinestimulates the body's immune system to recognize the agent as a foreigninvader, generate cellular immune responses and antibody (humoral)immune responses against it, and thereby inhibit or destroy it, anddevelop a memory of it. The vaccine-induced memory enables the immunesystem to act quickly to protect the body from any of these agents thatit later encounters. Vaccine production used in the art, e.g., antigenvaccine production, has several stages, including the generation ofantigens, antigen purification, in some cases inactivation of infectiousagents, and vaccine formulation. The first phase of this process is togenerate the antigen through culturing viruses in cell lines, growingbacteria in bioreactors, producing recombinant proteins derived fromviruses and bacteria in cell cultures, yeast or bacteria, or synthesizenucleic acids that express the antigens of interest (AOIs). The secondphase of the process is to purify the source of antigen, and in the caseof killed agent vaccines, to inactivate the virus, bacteria, orparasite. The third phase of the process is to create the actual vaccineformulation, which may include anything from simple dilution of the AOIin buffer, to the mixing of the AOI with adjuvants.

As demonstrated by the COVID-19 outbreak, vaccine development can be acostly, time consuming endeavor that is outpaced by fast-spreadinginfections. There is therefore a great need for the development of newvaccine production platform technologies that can be modularly adaptedto any new infectious agent. This need is also apparent for olderinfectious agents that mutate so rapidly that new vaccines are neededevery year, as is the case for flu. Furthermore, we need improvedtechnologies that generate vaccines that more closely mimic thephysicochemical state of the intact virus, which is often vesicular innature.

Consequently, there is a pressing need to develop new vaccines as wellas new approaches to combatting infectious diseases.

SUMMARY OF THE INVENTION

The present invention provides, in a first aspect, an engineered, ornon-naturally occurring, selectable marker (SM) protein, wherein the SMprotein comprises a destabilization domain (DD) appended to, oroperatively linked to, a SM protein, thereby providing a DD-tagged SMprotein, or a degron-tagged, or a non-naturally occurring, SM protein.In some embodiments, the engineered SM protein is for recombinantprotein expression. In some embodiments, said SM protein is a SM proteinfor, or that functions in, mammalian cells, e.g., human cells. In someembodiments, said DD is appended to the N-terminus of said SM protein,the C-terminus of said SM protein, or both the N-terminus and theC-terminus of said SM protein. In some embodiments, said DD is appendedto the N-terminus of said SM protein. In some embodiments, said SMprotein is a dominant SM protein. In some embodiments, said do SMprotein confers resistance to zeocin, puromycin, hygromycin, G418,and/or blasticidin. In some embodiments, said SM protein that confersresistance to zeocin is BleoR. In some embodiments, said SM protein thatconfers resistance to puromycin is PuroR. In some embodiments, said SMprotein that confers resistance to hygromycin is HygR. In someembodiments, said SM protein that confers resistance to G418 is NeoR. Insome embodiments, said SM protein that confers resistance to blasticidinis BsdR. In some embodiments, said DD is derived from the human estrogenreceptor (ER50), thereby providing a SM protein operably connected tothe ER50(DD). In some embodiments, said SM protein operably connected tothe ER50(DD) is BleoR operably connected to the ER50(DD), i.e.,ER50BleoR. In some embodiments, said SM protein operably connected tothe ER50(DD) is PuroR operatively connected to the ER50(DD), i.e.,ER50PuroR. In some embodiments, said SM protein operably connected tothe ER50(DD) is HygR operatively connected to the ER50(DD), i.e.,ER50HygR. In some embodiments, said SM protein operably connected to theER50(DD) is NeoR operatively connected to the ER50(DD), i.e., ER50NeoR.In some embodiments, said SM protein operably connected to the ER50(DD)is BsdR operatively connected to the ER50(DD), i.e., ER50BsdR. In someembodiments, said DD is derived from the human estrogen receptor (ER50),thereby providing an ER50(DD)-tagged SM protein. In some embodiments,said ER50(DD)-tagged SM protein is ER50(DD)-tagged BleoR (ER50BleoR),ER50(DD)-tagged PuroR (ER50PuroR), ER50(DD)-tagged HygR (ER50HygR),ER50(DD)-tagged NeoR (ER50NeoR), or ER50(DD)-tagged BsdR (ER50BsdR). Insome embodiments, said DD is derived from the Escherichia colidihydrofolate reductase (ecDHFR), thereby providing an SM proteinoperatively connected to the ecDHFR(DD). In some embodiments, said SMprotein operably connected to the ecDHFR(DD) is BleoR operatively linkedto the ecDHFR(DD), i.e., ecDHFRBleoR. In some embodiments, said SMprotein operably connected to the ecDHFR(DD) is PuroR operatively linkedto the ecDHFR(DD), i.e., ecDHFRPuroR. In some embodiments, said SMprotein operably connected to the ecDHFR(DD) is HygR operatively linkedto the ecDHFR(DD), i.e., ecDHFRHygR. In some embodiments, said SMprotein operably connected to the ecDHFR(DD) is NeoR operatively linkedto the ecDHFR(DD), i.e., ecDHFRNeoR. In some embodiments, said SMprotein operably connected to the ecDHFR(DD) is BsdR operatively linkedto the ecDHFR(DD), i.e., ecDHFRBsdR. In some embodiments, said DD isderived from the Escherichia coli dihydrofolate reductase (ecDHFR),thereby providing an ecDHFR(DD)-tagged SM protein. In some embodiments,said ecDHFR(DD)-tagged SM protein is ecDHFR(DD)-tagged BleoR(ecDHFRBleoR), ecDHFR(DD)-tagged PuroR (ecDHFRPuroR), ecDHFR(DD)-taggedHygR (ecDHFRHygR), ecDHFR(DD)-tagged NeoR (ecDHFRNeoR), orecDHFR(DD)-tagged BsdR (ecDHFRBsdR). In some embodiments, the engineeredSM protein further comprises an altered amino acid sequence resultingfrom a frameshift mutation within a nucleotide sequence that encodes thelast about 10, 20, 30, 40, or 50 amino acids at the 3′ end of theDD-tagged SM.

The present invention provides a nucleic acid, the nucleotide sequenceof which encodes the engineered SM protein according to the presentinvention. In some embodiments, said nucleic acid is an isolated nucleicacid.

The present invention provides an expression vector comprising a nucleicacid, the nucleotide sequence of which encodes a selectable marker (SM)protein and an operably linked recombinant protein of interest (POI),wherein the nucleic acid is operably linked to an expression controlsequence. In some embodiments, the SM protein is unstable and/ordegraded. In some embodiments, the SM protein selects for high-levelexpression of the linked recombinant POI. In some embodiments, saidexpression control sequence is a promoter. In some embodiments, saidpromoter is a cytomegalovirus (CMV) promoter. In some embodiments, saidnucleic acid comprises an open reading frame (ORF), e.g., a bicistronicORF, that encodes (a) the POI, followed by (b) a self-cleaving peptidewhich can induce ribosomal skipping during translation, and (c) the SMprotein. In some embodiments, said self-cleaving peptide which caninduce ribosomal skipping during translation is an about 18-22 aminoacid-long peptide. In some embodiments, said about 18-22 amino acid-longpeptide which can induce ribosomal skipping during translation is a 2aself-cleaving peptide. In some embodiments, said 2a self-cleavingpeptide is p2a, e2a, f2a or t2a. In some embodiments, said 2aself-cleaving peptide is a viral 2a peptide. In some embodiments, saidSM protein is PuroR2, having the amino acid sequence according to SEQ IDNO:1 (Table A), or a protein or a polypeptide sharing or having 60% orgreater amino acid sequence identity with, or having at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97% 98%, or 99% amino acid sequence identitywith, SEQ ID NO:1. In some embodiments, said SM protein is HygR2, havingthe amino acid sequence according to SEQ ID NO:2 (Table A), or a proteinor a polypeptide sharing or having 60% or greater amino acid sequenceidentity with, or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%98%, or 99% amino acid sequence identity with, SEQ ID NO:2. In someembodiments, said SM protein is NeoR2, having the amino acid sequenceaccording to SEQ ID NO:3 (Table A), or a protein or a polypeptidesharing or having 60% or greater amino acid sequence identity with, orhaving at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, or 99% aminoacid sequence identity with, SEQ ID NO:3. In some embodiments, said SMprotein is BsdR2, having the amino acid sequence according to SEQ IDNO:4 (Table A), or a protein or a polypeptide sharing or having 60% orgreater amino acid sequence identity with, or having at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97% 98%, or 99% amino acid sequence identitywith, SEQ ID NO:4. In some embodiments, said SM protein is BsdR5, havingthe amino acid sequence according to SEQ ID NO:5 (Table A), or a proteinor a polypeptide sharing or having 60% or greater amino acid sequenceidentity with, or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%98%, or 99% amino acid sequence identity with, SEQ ID NO:5.

TABLE A SEQ ID MTSNTPAVRPATRDDLPRALRTLQRAFADY NO: 1 PFTRHTIAADDHLARLHRFNELFVSRIGLD HGKVWVADDGDAVAVWTTPETADAGNVFAEIGPQFAEIAGDRADFSAQAEAAMGPHRPTE PVWFLGSVGVDPGRQGQGLGGAVIRPGLEAAEQAGVPAFLETSDERNVRFYERLGFEVTA DYPLPGGGPRTWAMTRKPGA SEQ IDMRKPIVSRSSLTATITRALGEVSDLTQITE NO: 2 GEESRAFSFRANGENYIVRINETVNGFNKDAYAYQRFATAALPIPEVVALGELDNGHAYC VSRRALGVTLQDLTRTELPAVVGPVASVLEAIASSTIGTASGYGPFDSQGRGAYATWRDF LTAIANPHQYKWNTLRHQVDVNRICLLLNEVLYLAEQCPEVRQLVHGDFGSNNVLTDGHR ITGVIDWSEAMVGDPLYDVANILFWRTWLECMEQQARFFEVHCADHLRPKERLRCYQLRI GLEEIYENALHGTADNVAWAINRCEEL SEQ IDMLEKDKFTTGSPAAWKVTLAGYRWIQQTIG NO: 3 CSEATVFRLDALGKPTLFVKTEPASPLSELQDEAARLRWLATVGLSCAQVLDSANEAGRD WLLLNVVPGENLLLASLDPVDKVTIMADALRRLHQLDPGTCPFDHRVRHRIERARDRIEA GLVDQDDLDEEHQGLEPAKLFARLRAHKPTTEDLVVTHGDACLPNIMVENGRFSGFIDCG RLGVADRYQDLALATRDIAEELGDEWIKPLLVQYGINDLDPDRTAFYRLLDEFY SEQ ID QITLTDKVTSKDQDELRLGLNAHNSKFFDV NO: 4DLIKPLGLFICDSQGKKLAGLTGTTTGNWL RIDLLWVSDSLRGQGTGSQLVLAAEKEARQRGCRFAQVDTASFQARPFYEKLGYHVRLTL GDYIHHHQRHYLTKIL SEQ IDMPLTTDETALVDAATSTITSIPISDTYSVA NO: 5 SAARSSDGRIFTGVNVFHFTGGPCAELVVLGCAAAAGATHLTHIVAVGNENRGIISPCGR CRQTLIDLHPGIKVVVLDRGEPRAVAVEEL LPFAYLVD

In some embodiments of the invention, said SM protein is the engineeredSM protein according to the present invention. In some embodiments, saidPOI is a transcriptional activator (TA). In some embodiments, saidtranscriptional activator is a reverse tetracycline transcriptionalactivator (rtTA). In some embodiments, said rtTA is rtTAv16 orrtTAv16/G72P. In some embodiments, said POI is an antigen of a pathogen.In some embodiments, said antigen of a pathogen is SARS-CoV-2 spike (S)protein, nucleocapsid (N) protein, membrane (M) protein, or envelope (E)protein. In some embodiments, said antigen of a pathogen is SARS-CoV-2spike (S) protein, nucleocapsid (N) protein, membrane (M) protein,envelope (E) protein, orf3a-encoded protein, and/or orf7a-encodedprotein. In one aspect, the expression vector POI comprises a plasmamembrane anchor and/or an oligomerization domain.

The present invention provides, in another aspect, PuroR2 gene whichencodes a GNAT family N-acetyltransferase protein from Streptomycesrimosus (WP_125058166.1), as well genes, codon optimized or otherwise,that encode a protein or a polypeptide sharing or having 60% or greateramino acid sequence identity with, or having at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97% 98%, or 99% amino acid sequence identity with,SEQ ID NO:1.

The present invention provides, in another aspect, HygR2 gene whichencodes an aminoglycoside-O-phosphotransferase protein from Ochrobactrumcytisi (WP_071631649.1), as well genes, codon optimized or otherwise,that encode a protein or a polypeptide sharing or having 60% or greateramino acid sequence identity with, or having at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97% 98%, or 99% amino acid sequence identity with,SEQ ID NO:2.

The present invention provides, in another aspect, NeoR2 gene whichencodes an aminoglycoside-O-phosphotransferase protein from Nitrosomonasoligotropha (WP_107804178.1), as well genes, codon optimized orotherwise, that encode a protein or a polypeptide sharing or having 60%or greater amino acid sequence identity with, or having at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, or 99% amino acid sequenceidentity with, SEQ ID NO:3.

The present invention provides, in another aspect, a selectable marker(SM) protein comprising C-terminal 17 amino acids of a 2a self-cleavingpeptide, wherein said C-terminal 17 amino acids of a 2a self-cleavingpeptide is tagged to the C-terminus of the SM.

The present invention provides, in another aspect, a protein of interest(POI) comprising an N-terminal proline of a 2a self-cleaving peptide,wherein said N-terminal proline of a 2a self-cleaving peptide is taggedto the N-terminus of the POI.

The present invention provides, in another aspect, BsdR2 gene whichencodes a GNAT family N-acetyltransferase from Pantoea ananatis(WP_024470972.1), as well genes, codon optimized or otherwise, thatencode a protein or a polypeptide sharing or having 60% or greater aminoacid sequence identity with, or having at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97% 98%, or 99% amino acid sequence identity with, SEQ IDNO:4.

The present invention provides, in another aspect, BsdR5 gene whichencodes a small molecules deaminase enzyme from Aspergillus udagawae(WP_024470972.1), or genes that encode a protein or a polypeptidesharing or having 60% or greater amino acid sequence identity with, orhaving at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, or 99% aminoacid sequence identity with, SEQ ID NO:5.

The present invention provides a cell comprising the expression vectoraccording to the present invention.

The present invention provides a cultured cell line comprising theexpression vector according to the present invention, wherein the cellsin the cultured cell line are selected by selection, or culturing, in aselector-containing media. In some embodiments, said selector-containingmedia is an antibiotic-containing media. In some embodiments, saidantibiotic-containing media contains zeocin, puromycin, hygromycin,G418, and/or blasticidin.

The present invention provides a method of making a cell line comprisingculturing cells transfected with the expression vector according to thepresent invention with a selector for the selectable marker. In someembodiments, said selector is an antibiotic. In some embodiments, saidantibiotic is zeocin, puromycin, hygromycin, G418, or blasticidin. Inone aspect the celltured in the presence of a selector for theselectable marker.

The present invention provides a method of making extracellular vesicles(EVs) comprising culturing the cell line according to the invention,wherein the cell line produces EVs comprising one or more of the POIsand isolating the EVs produced. In some embodiments, said EVs areexosomes or microvesicles.

The present invention provides a cultured cell line comprising cellscomprising the expression vector according to the invention, wherein thecells in the cell line have less cell-to-cell variance in POI productioncompared to cells which are not transfected with the expression vectoraccording to the invention. In some embodiments, the parental cell lineis 293F (Ftet cell line). In some embodiments, the cultured cell lineaccording to the present aspect of the present invention is amixed-clone (Ftet1) cell line of the Ftet cell line comprising aplurality of the Ftet cells which are resistant to a selector. In someembodiments, said selector is an antibiotic. In some embodiments, saidantibiotic is zeocin, puromycin, hygromycin, G418, or blasticidin. Insome embodiments, the cultured cell line according to the present aspectof the present invention is a single cell clone (Ftet2) cell line of theFtet cell line comprising a single cell clone (SCC) exhibiting thehighest relative expression of the transcriptional activator.

The present invention provides an expression vector comprising: (a) anucleic acid the nucleotide sequence of which encodes a transposoncomprised of inverted terminal repeats (ITR-L and ITR-R elements) thatdefine the left and right ends of the transposon; (b) one or more genesof interest (GOIs) encoding one or more proteins of interest (POIs)inserted between the ITR-L and ITR-R elements, wherein the GOIs areoperably linked to expression control sequences; and (c) a nucleic acidthe nucleotide sequence of which encodes a transposase enzyme, whereinthe nucleic acid is located outside the transposon, and wherein thenucleic acid is operably linked to an expression control sequence. Insome embodiments, the expression vector according to the aspect of thepresent invention further comprises (d) a nucleic acid, the nucleotidesequence of which encodes a selectable marker (SM) protein, wherein thenucleic acid is operably linked to an expression control sequence. Insome embodiments, said SM protein selects for high-level expression ofthe GOIs. In some embodiments, said SM protein is an unstable and/ordegraded SM protein. In some embodiments, the nucleic acid of element(d) is inserted between the ITR-L and ITR-R elements. In someembodiments, said expression control sequences operably linked to theGOIs include a plurality of binding sites for binding to atranscriptional activator, whereby the number of the binding sites canbe altered to modulate the POI stoichiometry. In some embodiments, saidtranscriptional activator is reverse tetracycline transcriptionalactivator (rtTA). In some embodiments, said rtTA is rtTAv16 orrtTAv16/G72P. In some embodiments, said expression control sequence is apromoter. In some embodiments, said promoter of element (d) is anEFlalpha (or its short, intron-less form, EFS) promoter. In someembodiments, said expression control sequences of element (b) areinducible promoters, thereby providing an expression vector forinducible expression of GOIs (inducible expression vector). In someembodiments, said inducible promoters are induced by tetracycline ordoxycycline. In some embodiments, said inducible promoters are TRE3Gpromoters. In some embodiments, said GOIs encode SARS-CoV-2 spike (S)protein, nucleocapsid (N) protein, membrane (M) protein, and/or envelope(E) proteins. In some embodiments, said GOIs encode SARS-CoV-2 spike (S)protein, nucleocapsid (N) protein, membrane (M) protein, envelope (E)protein, orf3a-encoded protein, and/or orf7a-encoded protein. In someembodiments, said S protein is the Wuhan-1 strain S protein; afurin-blocked, trimer-stabilized form of the Wuhan-1 strain S protein;or the Wuhan-1 strain S protein with an amino acid change of D614G. Insome embodiments, said transposase enzyme is a Sleeping Beauty (SB)transposase enzyme. In some embodiments, said nucleic acid thenucleotide sequence of which encodes a transposase enzyme comprises agene configured to express an optimized version of the SB transposaseenzyme. In some embodiments, said gene configured to express anoptimized version of the SB transposase enzyme is RSV-SB100x-pAn. Insome embodiments, said SM protein is PuroR2. In some embodiments, saidSM protein is the SM protein according to the present invention.

The present invention provides the cultured cell line according to thepresent invention, wherein the cultured cell line is further transfectedwith the expression vector according to the ninth aspect of the presentinvention. In some embodiments, the cultured cell line is furthertransfected with two or more of the expression vectors of the presentinvention, wherein each of said two or more of the expression vectorsaccording to the ninth aspect of the present invention has a separateselectable marker.

The present invention provides a method of making a cell line comprisingtransfecting the cultured cell line according to the present inventionwith the expression vector according to the present invention.

The present invention provides a method of making a cell line comprisingtransfecting the cultured cell line according to according to thepresent invention with two or more of the expression vectors accordingto the present invention, wherein each of said two or more of theexpression vectors according to the present invention has a separateselectable marker.

The present invention provides an extracellular vesicle-basedtherapeutic or prophylactic composition comprising a plurality ofextracellular vesicles (EVs) derived from the cultured cell lineaccording to the present invention, wherein said GOIs encode one or moretherapeutic or prophylactic proteins of interest (POIs). In someembodiments, said one or more therapeutic or prophylactic POIs areSARS-CoV-2 spike (S) protein, nucleocapsid (N) protein, membrane (M)protein, and/or envelope (E) proteins. In some embodiments, said one ormore therapeutic or prophylactic POIs are SARS-CoV-2 spike (S) protein,nucleocapsid (N) protein, membrane (M) protein, envelope (E) protein,orf3a-encoded protein, and/or orf7a-encoded protein. In someembodiments, the EV-based therapeutic or prophylactic compositionaccording to the present aspect of the present invention furthercomprises a physiologically acceptable excipient and/or adjuvant.

The present invention provides an expression vector comprising: (a) anucleic acid the nucleotide sequence of which encodes a first transposoncomprised of inverted terminal repeats (ITR-L and ITR-R elements) thatdefine the left and right ends of the first transposon; (b) one or moregenes of interest (GOIs) encoding one or more proteins of interest(POIs) inserted between the ITR-L and ITR-R elements of the firsttransposon, wherein the GOIs are operably linked to expression controlsequences; (c) a nucleic acid the nucleotide sequence of which encodes asecond transposon comprised of ITR-L and ITR-R elements that define theleft and right ends of the second transposon; (d) a nucleic acid thenucleotide sequence of which encodes a transcriptional activator,wherein the nucleic acid is inserted between the ITR-L and ITR-Relements of the second transposon; and (e) a nucleic acid the nucleotidesequence of which encodes a transposase enzyme, wherein the nucleic acidis located outside the first and second transposons, and wherein thenucleic acid is operably linked to an expression control sequence. Insome embodiments, the expression vector according to the present aspectof the present invention further comprises (f) a nucleic acid thenucleotide sequence of which encodes a selectable marker (SM) proteinthat selects for high-level expression of the GOIs. In some embodiments,said SM protein is an unstable and/or degraded SM protein. In someembodiments, the nucleic acid of element (f) is inserted between theITR-L and ITR-R elements of the first transposon. In some embodiments,said expression control sequences operably linked to the GOIs include aplurality of binding sites for binding to the transcriptional activator,whereby the number of the binding sites can be altered to modulate thePOI stoichiometry. In some embodiments, said transcriptional activatoris reverse tetracycline transcriptional activator (rtTA). In someembodiments, said rtTA is rtTAv16 or rtTAv16/G72P. In some embodiments,the expression control sequences of element (b) are inducible promoters,thereby providing an expression vector for inducible expression of GOIs(inducible expression vector). In some embodiments, said induciblepromoters are induced by tetracycline or doxycycline. In someembodiments, said inducible promoters are TRE3G promoters. In someembodiments, said GOIs encode SARS-CoV-2 spike (S) protein, nucleocapsid(N) protein, membrane (M) protein, and/or envelope (E) proteins. In someembodiments, said GOIs encode SARS-CoV-2 spike (S) protein, nucleocapsid(N) protein, membrane (M) protein, envelope (E) protein, orf3a-encodedprotein, and/or orf7a-encoded protein. In some embodiments, said Sprotein is the Wuhan-1 strain S protein; a furin-blocked,trimer-stabilized form of the Wuhan-1 strain S protein; or the Wuhan-1strain S protein with an amino acid change of D614G. In someembodiments, said transposase enzyme is a Sleeping Beauty (SB)transposase enzyme. In some embodiments, said nucleic acid thenucleotide sequence of which encodes a transposase enzyme comprises agene configured to express an optimized version of the SB transposaseenzyme. In some embodiments, said gene configured to express anoptimized version of the SB transposase enzyme is RSV-SB100x-pAn. Insome embodiments, said SM protein is PuroR2. In some embodiments, saidSM protein is the SM protein according to the present invention. In oneaspect, wherein the POI comprises a plasma membrane anchor and/or anoligomerization domain.

The present invention provides a cell comprising the expression vectoraccording to the present invention.

The present invention provides a cultured cell line comprising theexpression vector according to the present invention, wherein the cellsin the cultured cell line are selected by culturing in aselector-containing media. In some embodiments, said selector-containingmedia is an antibiotic-containing media. In some embodiments, saidantibiotic-containing media contains zeocin, puromycin, hygromycin,G418, and/or blasticidin.

The present invention provides a method of making a cell line comprisingculturing cells transfected with the expression vector according to anaspect of the present invention with a selector for the selectablemarker. In some embodiments, said selector is an antibiotic. In someembodiments, said antibiotic is zeocin, puromycin, hygromycin, G418, orblasticidin.

The present invention provides a method of making extracellular vesicles(EVs) comprising culturing a cell line according to the presentinvention, wherein the cell line produces EVs comprising one or more ofthe POIs and isolating the EVs produced. In some embodiments, said EVsare exosomes or microvesicles.

The present invention provides a cultured cell line comprising cellscomprising the expression vector according to the present invention,wherein the cells in the cell line have less cell-to-cell variance inPOI production compared to cells which are not transfected with theexpression vector according to the present invention. In someembodiments, the parental cell line is 293F (Ftet cell line). In someembodiments, the cultured cell line according to the present aspect ofthe present invention is a mixed-clone (Ftet1) cell line of the Ftetcell line comprising a plurality of the Ftet cells which are resistantto a selector. In some embodiments, said selector is an antibiotic. Insome embodiments, said antibiotic is zeocin, puromycin, hygromycin,G418, or blasticidin. In some embodiments, the cultured cell lineaccording to the present aspect of the present invention is a singlecell clone (Ftet2) cell line of the Ftet cell line comprising a singlecell clone (SCC) exhibiting the highest relative expression of thetranscriptional activator. culturing the cells in the presence of aselector for the selectable marker.

The present invention provides a pharmaceutical composition comprising aplurality of extracellular vesicles (EVs) derived from the cultured cellline according to the present invention, wherein said POI is atherapeutic or prophylactic POI. In some embodiments, said POI is atherapeutic or prophylactic POI. In some embodiments, said prophylacticPOI is an antigen of interest (AOI). In some embodiments, one or moreantigens of interest (AOIs) are displayed on the surface of the EVs. Insome embodiments, said AOIs are SARS-CoV-2 spike (S) protein,nucleocapsid (N) protein, membrane (M) protein, and/or envelope (E)proteins. In some embodiments, said AOIs are SARS-CoV-2 spike (S)protein, nucleocapsid (N) protein, membrane (M) protein, envelope (E)protein, orf3a-encoded protein, and/or orf7a-encoded protein. In someembodiments, said S protein is the Wuhan-1 strain S protein; afurin-blocked, trimer-stabilized form of the Wuhan-1 strain S protein;or the Wuhan-1 strain S protein with an amino acid change of D614G. Insome embodiments, the pharmaceutical composition according to thepresent aspect of the present invention further comprises aphysiologically acceptable excipient and/or adjuvant. In someembodiments, said EVs are exosomes or microvesicles.

The present invention provides a method of preventing a viral infectioncomprising administering to a subject susceptible to a viral infectionan immunogenically effective amount of the pharmaceutical compositionaccording to an aspect of the present invention.

The present invention provides an extracellular vesicle (EV)-basedantigen display composition comprising: an EV displaying one or moreantigens of interest (AOIs). In some embodiments, the EV-based antigendisplay composition according to the present aspect of the presentinvention is an EV-based antigen display vaccine. In some embodiments,the EV-based antigen display composition according to the present aspectof the present invention is an immune response stimulating composition.In some embodiments, said AOIs are displayed on the surface of the EVs.In some embodiments, said AOIs are SARS-CoV-2 spike (S) protein,nucleocapsid (N) protein, membrane (M) protein, and/or envelope (E)proteins. In some embodiments, said AOIs are SARS-CoV-2 spike (S)protein, nucleocapsid (N) protein, membrane (M) protein, envelope (E)protein, orf3a-encoded protein, and/or orf7a-encoded protein. In someembodiments, said S protein is the Wuhan-1 strain S protein; afurin-blocked, trimer-stabilized form of the Wuhan-1 strain S protein;or the Wuhan-1 strain S protein with an amino acid change of D614G. Insome embodiments, said EVs are exosomes or microvesicles. In one aspect,the EV-based antigen display composition is an immune responsestimulating composition. In one aspect, the EV-based antigen displaycomposition is a pharmaceutical composition including a physiologicallyacceptable excipient and/or adjuvant. In one aspect, the EV-basedantigen display composition is an immune response stimulatingcomposition.

In one aspect, the invention provides a composition comprisingextracellular vesicles (EVs) containing, as cargo (e.g., internally oron their surface) a protein of interest. For example, the protein ofinterest comprises SARS-CoV-2 spike (S) protein, nucleocapsid (N)protein, membrane (M) protein, envelope (E) protein, orf3a-encodedprotein, and/or orf7a-encoded protein. In another aspect, the protein ofinterest comprises an EV-trafficking element. Further, a composition ofthe invention can be formulated for administration to an animal (e.g., ahuman) subject via an oral route, a sublingual route, a buccal route, arectal route, a topical route, a transdermal route, injection,inhalation or a pulmonary route.

The present invention provides a pharmaceutical composition comprisingthe EV-based antigen display composition according to the presentinvention, and further comprising a physiologically acceptable excipientand/or adjuvant.

The present invention provides a method of preventing a viral infectioncomprising administering to a subject susceptible to a viral infectionan immunogenically effective amount of the EV-based antigen displaycomposition according to the present invention, or the pharmaceuticalcomposition according to the invention.

The present invention provides a composition comprising a population ofextracellular vesicles comprising a protein of interest (POI), whereinthe EVs in the population have higher POI expression compared with an EVpopulation made from cells not transfected with a selectable marker andselected for the selected marker, as disclosed herein, or have a lowerEV-to-EV variation (e.g., at least 25%, 50%, 75%, 80% or 90% lessvariation) in POI expression compared with an EV population made fromcells not transfected with a selectable marker and selected from theselected marker, as disclosed herein.

In one embodiment, the invention provides a composition includingextracellular vesicles (EVs) containing, as cargo (e.g., internally oron their surface) a protein of interest (POI). For example, a POI mayinclude SARS-CoV-2 spike (S) protein, nucleocapsid (N) protein, membrane(M) protein, envelope (E) protein, orf3a-encoded protein, and/ororf7a-encoded protein. In one aspect, the protein of interest comprisesan EV-trafficking element. In another aspect, the composition isformulated for administration to an animal (e.g., a human) subject viaan oral route, a sublingual route, a buccal route, a rectal route, atopical route, a transdermal route, injection, inhalation or a pulmonaryroute.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1I shows the transgene expression profiles of HEK293 cell linesarising from two-gene and bicistronic selections. (FIG. 1A) Line diagramshowing the NeoR and 3×NLS-tdTomato-2a-BsdR transgenes of the plasmidpJM825. (FIGS. 1B, 1C) Flow cytometry scatter plots of HEK293 cells thathad been transfected with pJM825 and then selected in (FIG. 1B) G418 or(FIG. 1C) blasticidin for 4 weeks. Numbers of cells are shown on they-axis while relative fluorescent brightness (arbitrary units (a.u.)) isshown on the x-axis (log scale). R3 shows the experimentally determinedbackground fluorescence of HEK293 control cells, whereas R4 denotes redfluorescence above background. (FIGS. 1D-1I) Fluorescence micrographs ofDAPI-stained (FIGS. 1D-F) HEK293/pJM825/G418-resistant cells and (FIGS.1G-1I) HEK293/pJM825/blasticidin-resistant cells, showing (FIGS. 1D, 1G)3×NLS-tdTomato, (FIGS. 1E, 1H) DAPI, and (FIGS. 1F, 1I) merged images.Bar, 100 μm. These experiments were performed in triplicate.

FIGS. 2A-2G shows the effect of selectable marker on linked expressionof 3×NLS-tdTomato. (FIG. 2A) Line diagram of transgenes encoding3×NLS-tdTomato, the viral p2a peptide, and the NeoR, BsdR, HygR, PuroR,and BleoR selectable markers (not to scale). Scatter plots of flowcytometric analyses of (FIG. 2B) HEK293 cells or (FIGS. 2C-2G) HEK293cells transfected with plasmids encoding the above transgenes andselected for 4 weeks in media containing (FIG. 2C) G418, (FIG. 2D)blasticidin, (FIG. 2E) hygromycin, (FIG. 2F) puromycin, or (FIG. 2G)zeocin. Numbers of cells are shown on the y-axis while relativefluorescent brightness (arbitrary units (a.u.)) is shown on the x-axis(log scale). R7 shows the experimentally determined backgroundfluorescence of HEK293 cells, whereas R8 denotes red fluorescence due to3×NLS-OtdTomato expression. These experiments were performed twice.

FIGS. 3A-3G shows the effect of selectable marker on linked expressionof CD81-mNG. (FIG. 3A) Line diagram of transgenes encoding CD81mNG, theviral p2a peptide, and the NeoR, BsdR, HygR, PuroR, and BleoR selectablemarkers (not to scale). Scatter plots of flow cytometric analyses of(FIG. 3B) HEK293 cells or (FIGS. 3C-3G) HEK293 cells transfected withplasmids encoding the above transgenes and selected for 4 weeks in mediacontaining (FIG. 3C) G418, (FIG. 3D) blasticidin, (FIG. 3E) hygromycin,(FIG. 3F) puromycin, or (FIG. 3G) zeocin. Numbers of cells are shown onthe y-axis while relative fluorescent brightness (arbitrary units(a.u.)) is shown on the x-axis (log scale). R3 shows the experimentallydetermined background fluorescence of HEK293 cells, whereas R4 denotesgreen fluorescence due to CD81mNG expression. These experiments wereperformed twice.

FIGS. 4A-4O shows the fluorescence micrographs of HEK293 cellstransfected with CD81mNG-expressing transgenes. HEK293 cells transfectedwith the five transgenes described in FIG. 3A were selected for 4 weeksin (FIGS. 4A-4C) G418, (FIGS. 4D-4F) blasticidin, (FIGS. 4G-41 )hygromycin, (FIGS. 4J-4L) puromycin, or (FIGS. 4M-4O) zeocin,respectfully. Each of these five cell lines were then grown overnight onsterile cover glasses, fixed, stained with DAPI. Images show (FIGS. 4A,4D, 4G, 4J, 4M) mNeonGreen fluorescence, (FIGS. 4B, 4E, 4H, 4K, 4N) DAPIfluorescence, and (FIGS. 4C, 4F, 4I, 4L, 4O) the merge of the two. Bar,100 μm. These experiments were performed in triplicate.

FIGS. 5A-5B shows the immunoblot analysis of HEK293 cells expressingCD81mNG. HEK293 cells transfected with the five transgenes described inFIG. 3A were selected for 4 weeks in G418, blasticidin, hygromycin,puromycin, or zeocin, respectfully. (FIG. 5A) Immunoblot analysis ofcell lysates probed using antibodies specific for (upper panel) the p2atag and (lower panel) actin. (FIG. 5B) Bar graphs show (upper graph)anti-2a signal intensity and (lower graph) anti-2a/actin signal ratiofor each polyclonal cell line. MW markers are, from top, 75 kDa, 50 kDa,37 kDa, and 25 kDa. This experiment was repeated three times.

FIGS. 6A-6D shows the size distribution profiles of exosomes released bytransgenic 293F cells. Exosomes were collected from the tissue culturesupernatants of (FIGS. 6A, 6B) 293F/pC-CD81mNG-2a-PuroR and (FIGS. 6C,6D) 293F/pC-CD81mNG-2a-BleoR cell lines and assayed by nanoparticletracking analysis. (FIGS. 6A, 6B) Scatter plots of exosome concentrationand size for (FIG. 6A) all 293F/pC-CD81mNG-2a-PuroR-derived exosomes and(FIG. 6B) green fluorescent 293F/pC-CD81mNG-2a-PuroR-derived exosomes.(FIGS. 6C, 6D) Scatter plots of exosome concentration and size for (FIG.6C) all 293F/pC-CD81mNG-2a-BleoR-derived exosomes and (FIG. 6D) greenfluorescent 293F/pC-CD81mNG-2a-BleoR-derived exosomes. These experimentswere performed once.

FIGS. 7A-7P shows the effect of transcriptional control elements andmode of transgene delivery on CD81mNG expression. (FIGS. 7A, 7B) Linediagrams of plasmid, Sleeping Beauty transposon, EBV-based episome, andlentiviral vectors carrying the (FIG. 7A) CMV-CD81mNG-2a-Puro and (FIG.7B) SFFV LTR-CD81mNG-2a-Puro transgenes. (FIGS. 7C-7J) HEK293 cells weretransfected or transduced with each of these vectors, selected inpuromycin, grown in selective media for 4 weeks, and assayed formNeonGreen fluorescence by flow cytometry. Numbers of cells are shown onthe y-axis while relative fluorescent brightness (arbitrary units(a.u.)) is shown on the x-axis (log scale). (FIGS. 7K-7P) HEK293 cellswere transfected with the six plasmid vectors shown (FIGS. 7A, 7B),grown for two days in normal media and assayed for mNeonGreenfluorescence by flow cytometry. Numbers of cells are shown on the y-axiswhile relative fluorescent brightness (arbitrary units (a.u.)) is shownon the x-axis (log scale). R7 shows the experimentally determinedbackground fluorescence of HEK293 control cells, whereas R8 denotesgreen fluorescence above background. These experiments were performedtwice.

FIGS. 8A-8O shows the fluorescence micrographs of African green monkeykidney COS7 cell lines carrying CD81mNG-expressing transgenes. COS7cells transfected with the five transgenes described in FIG. 3A wereselected for 4 weeks in (FIGS. 8A-8C) G418, (FIGS. 8D-8F) blasticidin,(FIGS. 8G-81 ) hygromycin, (FIGS. 8J-8L) puromycin, or (FIGS. 8M-80 )zeocin, respectfully. Each of these five cell lines were then grownovernight on sterile cover glasses, fixed, stained with DAPI. Imagesshow (FIGS. 8A, 8D, 8G, 8J, 8M) mNeonGreen fluorescence, (FIGS. 8B, 8E,8H, 8K, 8N) DAPI fluorescence, and (FIGS. 8C, 8F, 8I, 8L, 8O) the mergeof the two. Bar, 100 m. These images were selected from three technicalreplicates of the experiment.

FIG. 9 shows the line diagrams of non-replicating and Sleeping Beautyexpression vectors. The top two lines show the DNA sequence of thepolylinkers common to all p2a-containing and all pl-designated vectors.The linear plasmid maps depict the relative positions of major designelements of the circular plasmids created by the present inventors, withthe pC plasmids showing non-replicating vectors with the CMVtranscriptional control sequences, the pS plasmids showing thenon-replicating vectors with the SFFV LTR, the pITRSB-C plasmids showingthe Sleeping Beauty vectors with the CMV transcriptional controlelements, and the pITRSB-S showing the Sleeping Beauty vectors with theSFFV LTR.

FIG. 10 shows the line diagrams of lentiviral and replicating expressionvectors. The top two lines show the DNA sequence of the polylinkerscommon to all p2a-containing and all pl-designated vectors. The linearplasmid maps depict the relative positions of major design elements ofthe circular plasmids created by the present inventors, with thepLenti-C plasmids showing the structure of lentiviral provirusescarrying CMV transcriptional control sequences, the pLenti-S plasmidsshowing the structure of lentiviral proviruses carrying the SFFV LTR,and the pREP-C plasmids showing the structure of replicating vectorscarrying the CMV transcriptional control sequences.

FIG. 11 shows the line diagram of Sleeping Beauty vectors for testingeffects of selectable marker on linked recombinant protein (mCherry)expression.

FIG. 12 shows the mean mCherry fluorescence of selected cell lines.HEK293 cell lines transfected with the vectors described in FIG. 11 wereassayed for mCherry fluorescence by flow cytometry.

FIG. 13 shows the BlastP alignment of (upper line) PuroR2 proteinsequence and the (lower line) PuroR protein sequence.

FIG. 14 shows the map of plasmid pITRSB, the Sleeping Beauty donorplasmid developed by the present inventors.

FIG. 15 shows the map of plasmid pS179, an SB vector designed to delivertwo genes, one encoding PuroR and the other encoding mCherry.

FIG. 16 shows fluorescence brightness of Ftet1 cells (grey) andpuromycin-resistant Ftet1/pS179 cells (purple line).

FIG. 17 shows the map of plasmid pS147, a small plasmid with a singlegene. Promoter: CMV (inducible by prostratin). Predicted products:rtTAv16-2a protein (the 2a system tags the C-terminus of the upstreamprotein with 17 amino acids, to which we have an antibody);proline-BleoR (the downstream protein contains an N-terminal proline(the 18th amino acid of the p2a peptide) followed by the BleoR proteinsequence, which confers resistance to zeocin by binding it in a 1:1molar ratio.

FIG. 18 shows the immunofluorescence micrographs of 293F cells stainedwith (green channel) rabbit polyclonal anti-2a antibody andAlexa488-coupled goat anti-rabbit secondary antibody and (blue channel)Hoechst dye to label the nuclei. Images were collected using constantlamp brightness and exposure time, allowing 2a protein expression to beassessed on the basis of the green:blue staining ratio in these images.Note the absence of staining for 293F cells, establishing thespecificity of the assay.

FIG. 19 shows the map of plasmid pS180, for validation of the Tet-Onderivatives of 293F cells using a puromycin-resistant Sleeping Beautytransposon also carrying a Tet-inducible mCherry transgene.

FIG. 20 shows the flow cytometry measurements of mCherry expression inFtet1/pS180 cells +/−doxycycline.

FIG. 21 shows the flow cytometry measurements of mCherry expression inFtet1/pS180 cells +/−prostratin.

FIG. 22 shows the flow cytometry measurements of mCherry levels inFtet1/pS180 cells +/−prostratin, +/−doxycycline.

FIG. 23 shows the flow cytometry cytometry measurements of mCherryexpression in Ftet2 cells selected with the PuroR marker in either 1ug/ml puro or 3 ug/ml puro, or the ER50PuroR marker selected with 1ug/ml puro in the presence of 5 uM 4OHT.

FIG. 24 shows the flow cytometry measurements of mCherry expression inFtet2 cells selected with NeoR or ER50NeoR markers.

FIG. 25 shows the flow cytometry measurements of mCherry expression inFtet2 cells selected with BsdR or ER50BsdR markers.

FIG. 26 shows the map of plasmid ps226.

FIG. 27 shows the immunoblots of 293F/pS147/pS225 and 293F/pS147/pS226.

FIG. 28 shows the negative stain electron microscopy of VLPspreparations purified from the TC SN of 293F/pS147/pS226 cell culturesafter three days incubation in Freestyle media supplemented withdoxycycline.

FIG. 29 shows the study design for 293F/pS226 VLPs Immunization.

FIG. 30 shows the VLP antibody response (S-protein IgM): ELISA 14 daysversus 28 days.

FIG. 31 shows the VLP antibody response (S-protein IgG): ELISA 14 daysversus 28 days.

FIG. 32 shows the VLP antibody response (N-protein IgM): ELISA 14 daysversus 28 days.

FIG. 33 shows the VLP antibody response (N-protein IgG): ELISA 14 daysversus 28 days.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural references unless the contextclearly dictates otherwise. Thus, for example, references to “themethod” includes one or more methods, and/or steps of the type describedherein which will become apparent to those persons skilled in the artupon reading this disclosure and so forth.

The terms “treat”, “therapeutic”, “prophylactic” and “prevent” are notintended to be absolute terms. Treatment, prevention and prophylaxis canrefer to any delay in onset, amelioration of symptoms, improvement inpatient survival, increase in survival time or rate, etc. Treatment,prevention, and prophylaxis can be complete or partial. The term“prophylactic” means not only “prevent”, but also minimize illness anddisease. For example, a “prophylactic” agent can be administered tosubject to prevent infection, or to minimize the extent of illness anddisease caused by such infection. The effect of treatment can becompared to an individual or pool of individuals not receiving thetreatment, or to the same patient prior to treatment or at a differenttime during treatment. In some aspects, the severity of disease isreduced by at least 10%, as compared, e.g., to the individual beforeadministration or to a control individual not undergoing treatment. Insome aspects, the severity of disease is reduced by at least 25%, 50%,75%, 80%, or 90%, or in some cases, no longer detectable using standarddiagnostic techniques.

A treatment can be considered “effective,” as used herein, if one ormore of the signs or symptoms of a condition described herein arealtered in a beneficial manner, other clinically accepted symptoms areimproved, or even ameliorated, or a desired response is induced e.g., byat least 2%, 3%, 4%, 5%, 10%, or more, following treatment according tothe methods described herein. Efficacy can be assessed, for example, bymeasuring a marker, indicator, symptom, and/or the incidence of acondition treated according to the methods described herein or any othermeasurable parameter appropriate. Efficacy can also be measured by afailure of an individual to worsen as assessed by hospitalization, orneed for medical interventions (e.g., progression of the disease ishalted). Treatment includes any treatment of a disease in an individualor an animal (some non-limiting examples include a human or an animal)and includes: (1) inhibiting the disease, e.g., preventing a worseningof symptoms (e.g. pain or inflammation); or (2) relieving the severityof the disease, e.g., causing regression of symptoms. An effectiveamount for the treatment of a disease means that amount which, whenadministered to a subject in need thereof, is sufficient to result ineffective treatment as that term is defined herein, for that disease.Efficacy of an agent can be determined by assessing physical indicatorsof a condition or desired response. One skilled in the art can monitorefficacy of administration and/or treatment by measuring any one of suchparameters, or any combination of parameters.

The term “effective amount” as used herein refers to the amount of acomposition or an agent needed to alleviate at least one or more symptomof the disease or disorder, and relates to a sufficient amount oftherapeutic composition to provide the desired effect. The term“therapeutically effective amount” refers to an amount of a compositionor therapeutic agent that is sufficient to provide a particular effectwhen administered to a typical subject. An effective amount as usedherein, in various contexts, can include an amount sufficient to delaythe development of a symptom of the disease, alter the course of asymptom disease (for example but not limited to, slowing the progressionof a symptom of the disease), or reverse a symptom of the disease. Forexample, for the given parameter, a therapeutically effective amountwill show an increase or decrease of therapeutic effect at least any of5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%.Therapeutic efficacy can also be expressed as “-fold” increase ordecrease. For example, a therapeutically effective amount can have atleast any of a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effect over acontrol. The therapeutically effective amount may be administered in oneor more doses of the therapeutic agent. The therapeutically effectiveamount may be administered in a single administration, or over a periodof time in a plurality of doses.

“Administering” as used herein can include any suitable routes ofadministering a therapeutic agent or composition as disclosed herein.Suitable routes of administration include, without limitation, oral,parenteral, intravenous, intramuscular, subcutaneous, transdermal,airway (aerosol), pulmonary, cutaneous, injection or topicaladministration. Administration can be local or systemic.

As used herein, the term “pharmaceutically acceptable” refers to acarrier that is compatible with the other ingredients of the formulationand not deleterious to the recipient thereof. The term is usedsynonymously with “physiologically acceptable” and “pharmacologicallyacceptable”. A pharmaceutical composition will generally comprise agentsfor buffering and preservation in storage, and can include buffers andcarriers for appropriate delivery, depending on the route ofadministration. The phrase “pharmaceutically acceptable” is employedherein to refer to those compounds, materials, compositions, and/ordosage forms which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of human beings and animalswithout excessive toxicity, irritation, allergic response, or otherproblem or complication, commensurate with a reasonable benefit/riskratio.

The terms “dose” and “dosage” are used interchangeably herein. A doserefers to the amount of active ingredient given to an individual at eachadministration. For the present invention, the dose can refer to theconcentration of the extracellular vesicles or associated components,e.g., the amount of therapeutic agent or dosage of radiolabel. The dosewill vary depending on a number of factors, including frequency ofadministration; size and tolerance of the individual; severity of thecondition; risk of side effects; the route of administration; and theimaging modality of the detectable moiety (if present). One of skill inthe art will recognize that the dose can be modified depending on theabove factors or based on therapeutic progress. The term “dosage form”refers to the particular format of the pharmaceutical, and depends onthe route of administration. For example, a dosage form can be in aliquid, e.g., a saline solution for injection.

“Subject,” “patient,” “individual” and like terms are usedinterchangeably and refer to, except where indicated, mammals such ashumans and non-human primates, as well as rabbits, rats, mice, goats,pigs, and other mammalian species. The term does not necessarilyindicate that the subject has been diagnosed with a particular disease,but typically refers to an individual under medical supervision. Apatient can be an individual that is seeking treatment, monitoring,adjustment or modification of an existing therapeutic regimen, etc.

As used herein, the following meanings apply unless otherwise specified.The word “may” is used in a permissive sense (i.e., meaning having thepotential to), rather than the mandatory sense (i.e., meaning must). Thewords “include”, “including”, and “includes” and the like meanincluding, but not limited to. The singular forms “a,” “an,” and “the”include plural referents. Thus, for example, reference to “an element”includes a combination of two or more elements, notwithstanding use ofother terms and phrases for one or more elements, such as “one or more.”The term “or” is, unless indicated otherwise, non-exclusive, i.e.,encompassing both “and” and “or.” The term “any of” between a modifierand a sequence means that the modifier modifies each member of thesequence. So, for example, the phrase “at least any of 1, 2 or 3” means“at least 1, at least 2 or at least 3”. The phrase “at least one”includes “a plurality”.

Definitions of common terms in cell biology and molecular biology can befound in “The Merck Manual of Diagnosis and Therapy”, 19th Edition,published by Merck Research Laboratories, 2006 (ISBN 0-91 1910-19-0);Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Biology,published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); BenjaminLewin, Genes X, published by Jones & Bartlett Publishing, 2009 (ISBN-10:0763766321); Kendrew et al. (eds.), Molecular Biology and Biotechnology:a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995(ISBN 1-56081-569-8) and Current Protocols in Protein Sciences 2009,Wiley Intersciences, Coligan et al., eds.

The term “selectable marker” (SM) refers to a gene introduced into acell that confers a trait suitable for artificial selection. Examples ofselectable markers includes antibiotic resistance markers, such as BleoR(zeocin resistance), PuroR (puromycin resistance), HygR (hygromycinresistance), NeoR (G418 resistance), BsdR (blasticidin resistance), AmpR(ampicillin resistance), TetR (tetracycline resistance), and KanR(kanamycin resistance).

The term “dominant selectable marker” refers to a protein that isencoded by a conditionally dominant gene introduced into a cell thatconfers an ability to grow in the presence of applied selective agentsthat are normally toxic to cells or inhibitory to cell growth, such asantibiotics.

The term “destabilization domain” (DD) refers to a protein, polypeptideor amino acid sequence that modulates the stability of a protein whenoperably connected to, linked to, or fused to (e.g., as a fusioncomponent of), the protein. For example, the term “destabilizationdomain” (DD), refers to a protein domain that is unstable and degradedin the absence of ligand, but whose stability is rescued by binding to ahigh affinity cell-permeable ligand. Destabilization domains (DDs) canbe fused or linked to a target protein and can convey its destabilizingproperty to the protein of interest, causing protein degradation. DDsrender the attached protein of interest unstable in the absence of aDD-binding ligand such that the protein is rapidly degraded by theubiquitin-proteasome system of the cell. However, when a specific smallmolecule ligand binds its intended DD as a ligand binding partner, theinstability is reversed and some level of protein function is restored.The conditional nature of DD stability allows a rapid and non-perturbingswitch from stable protein to unstable substrate for degradation. Such adestabilization domain may or may not require the interaction of anotherprotein for modulating stability of the protein. Non-limiting examplesof DDs include structurally unstable protein domains derived fromEscherichia coli dihydrofolate reductase (DHFR), as described in IwamotoM, Björklund T, Lundberg C, Kirik D, Wandless T J. Chem Biol. 2010;17:981-988, and the human estrogen receptor (ER50), as described inMiyazaki Y, Imoto H, Chen L C, Wandless T J. J Am Chem Soc. 2012;134:3942-3945, as well as Maji B, Moore C L, Zetsche B, Volz S E, ZhangF, Shoulders M D, Choudhary A. Multidimensional chemical control ofCRISPR-Cas9. Nat Chem Biol. 2017 January; 13(1):9-11, the contents ofwhich are each incorporated herein by reference in their entirety. Theterm “extracellular vesicle” (EV) refers to lipid bilayer-delimitedparticles that are naturally released from cells. EVs range in diameterfrom around 20-30 nanometers to about 10 microns or more. EVs cancomprise proteins, nucleic acids, lipids and metabolites from the cellsthat produced them. EVs include exosomes (about 50 to about 100 nm),microvesicles (about 100 to about 300 nm), ectosomes (about 50 to about1000 nm), apoptotic bodies (about 50 to about 5000 nm) and lipid-proteinaggregates of the same dimensions. In another example, thedestabilization domain can comprise KEN, Cyclin A, UFD domain/substrate,ubiquitin, PEST sequences, destruction boxes and hydrophobic stretchesof amino acids. Exemplary destabilization domains include ubiquitin andhomologs thereof, particularly those comprising mutations that prevent,or significantly reduce, the cleavage of ubiquitin multimers bya-NH-ubiquitin protein endoproteases.

The term “virus-like particle” or “VLP” is meant to refer to anysupramolecular nanoparticle (ranging from 20 to 100 nm) that comprisesenvelope and/or capsid viral proteins, but that is non-infectiousbecause it does not contain viral genetic material. VLPs are useful aspart of vaccine composition, as they contain repetitive, high densitydisplays of viral surface proteins presenting conformational viralepitopes that can elicit T cell and B cell immune responses. Since VLPscannot replicate, they provide a safer alternative to attenuatedviruses. EVs as described herein, which include molecules that resembleviruses but that are not infectious and non-replicative can be used asVLPs.

Extracellular vesicles and virus-like particles can be referred toherein as “delivery vehicles.” An extracellular vesicle can carry acargo, which can be a protein of interest (POI) or a nucleic acid ofinterest (NAOI). The cargo molecule can be present within the lumen ofthe delivery vehicle or on its surface. The protein of interest can be aprotein that is naturally produced by a cell that generates a deliveryvehicle, or it can be a recombinant protein, including a non-naturallyoccurring protein, such as a fusion protein. The POI can be a viralprotein, e.g., capable of eliciting an immune response. Nucleic acidsinclude, without limitation, DNA and RNA. RNA can be mRNA. Whendelivered to a target cell, mRNA may be expressed as protein andpresented on the cell surface to elicit an immune response. Nucleicacids are typically incorporated into EVs by contacting the EVs and thenucleic acid in the presence of a chemical lipofection reagent. Thechemical lipofection reagent can be a polycationic lipid. In someembodiments, the chemical lipofection reagent is an mRNA lipofectionreagent, or an mRNA transfection reagent, e.g., Lipofectamine®MessengerMAX™, Lipofectamine® 2000, Lipofectamine® 3000.

The nucleotide and amino acid sequences of the SARS-CoV-2 Wuhan-1 strainspike (S) protein, and the Wuhan-1 strain S protein with an amino acidchange of D614G are well known in the art, and are described in, e.g.,Plante, J. A., Liu, Y., Liu, J. et al. Spike mutation D614G altersSARS-CoV-2 fitness. Nature (2020), the contents of which are eachincorporated herein by reference in their entirety.

The term “nucleic acid” refers to polynucleotides such asdeoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Nucleic acidsinclude but are not limited to genomic DNA, cDNA, mRNA, iRNA, miRNA,tRNA, ncRNA, rRNA, and recombinantly produced and chemically synthesizedmolecules such as aptamers, plasmids, anti-sense DNA strands, shRNA,ribozymes, nucleic acids conjugated and oligonucleotides. According tothe invention, a nucleic acid may be present as a single-stranded ordouble-stranded and linear or covalently circularly closed molecule. Anucleic acid might be employed for introduction into, e.g., transfectionof, cells, e.g., in the form of RNA which can be prepared by in vitrotranscription from a DNA template. The RNA can moreover be modifiedbefore application by stabilizing sequences, capping, andpolyadenylation. Generally, nucleic acid can be extracted, isolated,amplified, or analyzed by a variety of techniques such as thosedescribed by, e.g., Green and Sambrook, Molecular Cloning: A LaboratoryManual (Fourth Edition), Cold Spring Harbor Laboratory Press, Woodbury,N.Y. 2,028 pages (2012).

A SARS-CoV-2 virion is approximately 50-200 nanometers in diameter. Likeother coronaviruses, SARS-CoV-2 has four structural proteins, known asthe S (spike), E (envelope), M (membrane), and N (nucleocapsid)proteins; the N protein holds the RNA genome, and the S, E, and Mproteins together create the complete viral envelope, which are theproteins of interest in regard to the present invention. The spikeprotein, S, which has been imaged at the atomic level using cryogenicelectron microscopy, is the protein responsible for allowing the virusto attach to and fuse with the membrane of a host cell. As used herein,the phrase SARS-CoV-2 structural “protein S, N, M, and/or E” refers tothe spike (S), nucleocapsid (N), membrane (M), and/or envelope (E)proteins, respectively, which are encoded by the nucleic acid sequencesof the invention, or by a codon-optimized oligonucleotide sequence,encoding each protein individually, or any combination of 2 or 3proteins, or a combination of all 4 proteins. When two or more nucleicacid sequences are included in a single vector or construct, they are inoperable linkage such that the each of the 2, 3, or 4 SARS-CoV-2structural proteins are properly encoded and expressed. Nucleic acidsequences encoding additional SARS-CoV-2 proteins, such as orfa ororfa/b polypeptides are also included in the nucleic acid sequences ofthe present invention. Such nucleic acid sequences may be incorporatedin a vector as described herein to provide a variation of these vectors.Cells transfected with a vector as described herein, may be transfectedwith a vector including a nucleic acid sequence encoding an additionalSARS-CoV-2 protein.

The term “vector”, “expression vector”, or “plasmid DNA” is used hereinto refer to a recombinant nucleic acid construct that is manipulated byhuman intervention. A recombinant nucleic acid construct can contain twoor more nucleotide sequences that are linked in a manner such that theproduct is not found in a cell in nature. For instance, the two or morenucleotide sequences can be operatively linked, such as one or moregenes encoding one or more proteins of interest, one or more proteintags, functional domains and the like. For example, the proteins ofinterest according to the invention can include SARS-CoV-2 structuralprotein S, N, M, and/or E. The expression vector of the invention caninclude regulatory elements controlling transcription generally derivedfrom mammalian, microbial, viral or insect genes, such as an origin ofreplication to confer the vector the ability to replicate in a host, anda selection gene encoding, e.g., a selectable marker (SM) protein, tofacilitate recognition of transformants may additionally beincorporated. Those of skill in the art can select a suitable regulatoryregion to be included in such a vector depending on the host cell usedto express the gene(s). For example, the expression vector can compriseone or more promoters, operably linked to the nucleic acid of interest,or a gene of interest (GOI), capable of facilitating transcription ofgenes in operable linkage with the promoter. Several types of promotersare well known in the art and suitable for use with the presentinvention. The promoter can be constitutive or inducible. ADDitionalregulatory elements that may be useful in vectors include, but are notlimited to, polyadenylation sequences, translation control sequences(e.g., an internal ribosome entry segment, IRES), enhancers, introns,and the like. Such elements may not be necessary, although they mayincrease expression by affecting transcription, stability of the mRNA,translational efficiency, or the like. Such elements can be included ina nucleic acid construct as desired to obtain optimal expression of thenucleic acids in the cell(s). Sufficient expression, however, maysometimes be obtained without such additional elements. Vectors also caninclude other elements. For example, a vector can include a nucleic acidthat encodes a signal peptide such that the encoded polypeptide isdirected to a particular cellular location (e.g., a signal secretionsequence to cause the protein to be secreted by the cell) or a nucleicacid that encodes a selectable marker. Non-limiting examples ofselectable markers include doxycycline, puromycin, adenosine deaminase(ADA), aminoglycoside phosphotransferase (neo, G418, APH), dihydrofolatereductase (DHFR), hygromycin-B-phosphotransferase, thymidine kinase(TK), and xanthin-guanine phosphoribosyltransferase (XGPRT). Suchmarkers are useful for selecting stable transformants in culture.Non-limiting examples of vectors suitable for use for the expression ofhigh levels of recombinant proteins of interest include those selectedfrom baculovirus, phage, plasmid, phagemid, cosmid, fosmid, bacterialartificial chromosome, viral DNA, Pl-based artificial chromosome, yeastplasmid, transposon, and yeast artificial chromosome. For example, theviral DNA vector can be selected from vaccinia, adenovirus, foul poxvirus, pseudorabies and a derivative of SV40. Non-limiting examples ofsuitable bacterial vectors include pQE70™, pQE60™, pQE-9™, pBLUESCRIPT™SK, pBLUESCRIPT™ KS, pTRC99a™, pKK223-3™, pDR540™, PAC™ and pRIT2T™.Non-limiting examples of suitable eukaryotic vectors include pWLNEO™,pXTI™, pSG5™, pSVK3™, pBPV™, pMSG™, and pSVLSV40™. Non-limiting examplesof suitable eukaryotic vectors include pWLNEO™, pXTI™, pSG5™, pSVK3™,pBPV™, pMSG™, and pSVLSV40™. One type of vector is a genomic integratedvector which can become integrated into the chromosomal DNA of the hostcell. Another type of vector is an episomal vector, e.g., a nucleic acidcapable of extra-chromosomal replication. Viral vectors includeadenovirus, adeno-associated virus (AAV), retroviruses, lentiviruses,vaccinia virus, measles viruses, herpes viruses, and bovine papillomavirus vectors (see, Kay et al., Proc. Natl. Acad. Sci. USA94:12744-12746 (1997) for a review of viral and non-viral vectors).Viral vectors can be modified so the native tropism and pathogenicity ofthe virus are altered or removed. The genome of a virus also can bemodified to increase its infectivity and to accommodate packaging of thenucleic acid encoding the polypeptide of interest.

The term “derived from” as in “A is derived from B” means that A isobtained from B in such a manner that A is not identical to B. Forinstance, if a destabilization domain (DD) is “derived from” theEscherichia coli dihydrofolate reductase (ecDHFR), that means thedegradation domain of the ecDHFR is obtained from ecDHFR, therebyproviding ecDHFR(DD). Accordingly, the term “ecDHFRBsdR” refers to BsdRwhich has been derivatized by appending the degradation domain of theecDHFR to the N-terminus of BsdR. The term “polycistronic” (e.g.,“bicistronic”) refers to a nucleic acid molecule, e.g., mRNA, which,upon translation, produces a plurality of polypeptides. A plurality ofpolypeptides can be produced by, for example, inclusion of a pluralityof open reading frames, or a single reading frame comprising aself-cleaving peptide, such as viral 2A peptide.

The term “self-cleaving peptide” refers to a peptide that mediatesribosome-skipping events during translation, producing independentpolypeptides from a single message. Examples of self-cleaving peptidesinclude peptides of the 2A peptide family, including: T2A—EGRGSLLTCGDVEENPGP (thosea asigna virus 2A); P2A—ATNFSLLKQAGDVEENPGP (porcineteschovirus-1 2A); E2A—QCTNYALLKLAGDVESNPGP (equine rhinitis A virus)and F2A—VKQTLNFDLLKLAGDVESNPGP (foot-and-mouth disease virus 18). Addingthe optional linker “GSG” (Gly-Ser-Gly) on the N-terminal of a 2Apeptide helps with efficiency.

The term “transgene” refers to a gene in a cell or organism that is notnative to the at cell or organism, typically incorporated naturally, orby any of a number of genetic engineering techniques.

The term “open reading frame” (ORF) refers to a nucleotide sequence,typically positioned between a start codon and a stop codon, that hasthe ability to be translated into a polypeptide.

The term “expression control sequence” refers to a nucleotide sequencethat regulates transcription and/or translation of a nucleotide sequenceoperatively linked thereto. Expression control sequences include, butare not limited to, promoters, enhancers, repressors (transcriptionregulatory sequences) and ribosome binding sites (translation regulatorysequences).

As used herein, a nucleotide sequence is “operably linked” with anexpression control sequence when the expression control sequencefunctions in a cell to regulate transcription of the nucleotidesequence. This includes promoting transcription of the nucleotidesequence through an interaction between a polymerase and a promoter.

The term “clone” refers to a group of identical cells that share acommon ancestry, e.g., they are derived from the same cell.

A variety of host cells are known in the art and suitable for proteinsexpression and extracellular vesicles production. Non-limiting examplesof typical cell used for transfection include, but are not limited to, abacterial cell, a eukaryotic cell, a yeast cell, an insect cell, or aplant cell. For example, human embryonic kidney 293 (HEK293), E. coli,Bacillus, Streptomyces, Pichia pastoris, Salmonella typhimurium,Drosophila S2, Spodoptera SJ9, CHO, COS (e.g. COS-7), 3T3-F442A, HeLa,HUVEC, HUAEC, NIH 3T3, Jurkat, 293, 293H, or 293F.

A variety of methods are known in the art and suitable for introductionof nucleic acid into a cell, including viral and non-viral mediatedtechniques. Non-limiting examples of typical non-viral mediatedtechniques includeelectroporation, calcium phosphate mediated transfer,nucleofection, sonoporation, heat shock, magnetofection, liposomemediated transfer, microinjection, microprojectile mediated transfer(nanoparticles), cationic polymer mediated transfer (DEAE-dextran,polyethylenimine, polyethylene glycol (PEG) and the like) or cellfusion. Other methods of transfection include, but are not limited to,proprietary transfection reagents such as LIPOFECTAMINE™, DOJINDOHILYMAX™, FUGENE™, JETPEI™, EFFECTENE™ and DREAMFECT™.

A pathogen, which can be a bacteria, virus, or any other microorganismthat can cause a disease in a subject, can elicit an immune response(i.e., an integrated bodily response to a pathogen antigen, which caninclude a cellular immune response and/or a humoral immune response) inthe subject. For example, upon contact and/or exposure to a pathogen, asubject may respond with an humoral immune response, characterized bythe production of antibody, specifically directed against one or morepathogen antigens.

As used herein the term “antibody” refers to immunoglobulin (Ig)molecules and immunologically active portions of immunoglobulinmolecules, i.e., molecules that contain an antigen-binding site thatspecifically binds an antigen. Antibodies are usually heterotetramericglycoproteins of about 150,000 Daltons, composed of two identical light(L) chains and two identical heavy (H) chains. The light chains from anyvertebrate species can be assigned to one of two clearly distinct types,called kappa (κ) and lambda (λ), based on the amino acid sequences oftheir constant domains. Depending on the amino acid sequence of theconstant domain of their heavy chains, immunoglobulins can be assignedto different classes. There are five major classes of immunoglobulins:IgA, IgD, IgE, IgG, and IgM, and several of these may be further dividedinto subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.The heavy-chain constant domains that correspond to the differentclasses of immunoglobulins are called a, 6, c, y, and μ, respectively.The subunit structures and three-dimensional configurations of differentclasses of immunoglobulins are well known. The antibody may have one ormore effector functions which refer to those biological activitiesattributable to the Fc region (a native sequence Fc region or amino acidsequence variant Fc region or any other modified Fc region) of anantibody. Non-limiting examples of antibody effector functions includeC1q binding; complement dependent cytotoxicity; Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor (BCR); andcross-presentation of antigens by antigen presenting cells or dendriticcells).

The term “neutralizing antibody” (Nab) refers an antibody that defends acell from a pathogen or infectious particle by neutralizing any effectit has biologically. Neutralization renders the particle no longerinfectious or pathogenic. Neutralizing antibodies are part of thehumoral response of the adaptive immune system against viruses,intracellular bacteria and microbial toxin. By binding specifically tosurface antigen on an infectious particle, neutralizing antibodiesprevent the particle from interacting with its host cells it mightinfect and destroy. Immunity due to neutralizing antibodies is alsoknown as sterilizing immunity, as the immune system eliminates theinfectious particle before any infection took place.

The term “antigen” refers to any substance that will elicit an immuneresponse. For instance, an antigen relates to any substance, preferablya peptide or protein, that reacts specifically with antibodies orT-lymphocytes (T cells). As used herein, the term “antigen” comprisesany molecule which comprises at least one epitope. For instance, anantigen is a molecule which, optionally after processing, induces animmune reaction. For instance, any suitable antigen may be used, whichis a candidate for an immune reaction, wherein the immune reaction maybe a cellular immune reaction. For instance, the antigen may bepresented by a cell, which results in an immune reaction against theantigen. For example, an antigen is a product which corresponds to or isderived from a naturally occurring antigen. Such antigens include, butare not limited to, SARS-CoV-2 structural proteins S, N, M, and E, andany variants or mutants thereof.

The terms “peptide”, “polypeptide” and “protein” are usedinterchangeably herein and refer to any chain of at least two aminoacids, linked by a covalent chemical bound. As used herein a peptide canrefer to the complete amino acid sequence coding for an entire proteinor to a portion thereof. A “protein coding sequence” or a sequence that“encodes” a particular polypeptide or peptide, is a nucleic acidsequence that is transcribed (in the case of DNA) and is translated (inthe case of mRNA) into a polypeptide in vitro or in vivo when placedunder the control of appropriate regulatory sequences. The boundaries ofthe coding sequence are determined by a start codon at the 5′ (amino)terminus and a translation stop codon at the 3′ (carboxyl) terminus. Acoding sequence can include, but is not limited to, cDNA fromprokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryoticor eukaryotic DNA, and even synthetic DNA sequences. A transcriptiontermination sequence will usually be located 3′ to the coding sequence.

The term “pharmaceutical composition” refers to a formulation comprisingan active ingredient, and optionally a pharmaceutically acceptablecarrier, diluent or excipient. The term “active ingredient” caninterchangeably refer to an “effective ingredient,” and is meant torefer to any agent that is capable of inducing a sought-after effectupon administration. By “pharmaceutically acceptable” it is meant thecarrier, diluent or excipient must be compatible with the otheringredients of the formulation and not deleterious to the recipientthereof, nor to the activity of the active ingredient of theformulation. Pharmaceutically acceptable carriers, excipients orstabilizers are well known in the art, for example Remington'sPharmaceutical Sciences, 16th edition, Osol, A. Ed. (1980).Pharmaceutically acceptable carriers, excipients, or stabilizers arenontoxic to recipients at the dosages and concentrations employed, andmay include buffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (for example, Zn-protein complexes); and/ornon-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol(PEG). Examples of carrier include, but are not limited to, liposome,nanoparticles, ointment, micelles, microsphere, microparticle, cream,emulsion, and gel. Examples of excipient include, but are not limitedto, anti-adherents such as magnesium stearate, binders such assaccharides and their derivatives (sucrose, lactose, starches,cellulose, sugar alcohols and the like) protein like gelatin andsynthetic polymers, lubricants such as talc and silica, andpreservatives such as antioxidants, vitamin A, vitamin E, vitamin C,retinyl palmitate, selenium, cysteine, methionine, citric acid, sodiumsulfate and parabens. Examples of diluent include, but are not limitedto, water, alcohol, saline solution, glycol, mineral oil and dimethylsulfoxide (DMSO).

The term “vaccine” relates to a pharmaceutical preparation(pharmaceutical composition) or product that upon administration inducesan immune response, e.g., a cellular immune response, which recognizesand attacks a pathogen or a diseased cell. The term “immune response”refers to an integrated bodily response to an antigen and refers to acellular immune response and/or a humoral immune response. The immuneresponse may be protective/preventive/prophylactic and/or therapeutic.

A “cellular immune response” can include a cellular response directed tocells characterized by presentation of an antigen with class I or classII MEW, or a humoral response directed to the production of antibodies.The cellular response relates to cells called T cells or T-lymphocyteswhich act as either “helpers” or “killers”. The helper T cells (alsotermed CD4+ T cells) play a central role by regulating the immuneresponse and the killer cells (also termed cytotoxic T cells, cytolyticT cells, CD8+ T cells or CTLs) kill diseased cells such as cancer cells,preventing the production of more diseased cells.

The terms “immunoreactive cell” “immune cells” or “immune effectorcells” relate to a cell which exerts effector functions during an immunereaction. An “immunoreactive cell” preferably is capable of binding anantigen or a cell characterized by presentation of an antigen or anantigen peptide derived from an antigen and mediating an immuneresponse. For example, such cells secrete cytokines and/or chemokines,secrete antibodies, recognize cancerous cells, and optionally eliminatesuch cells. For example, immunoreactive cells comprise T cells(cytotoxic T cells, helper T cells, tumor infiltrating T cells), Bcells, natural killer cells, neutrophils, macrophages, and dendriticcells.

The term “adjuvant” refers to a pharmacological or immunological agentthat modifies the effect of other agents. An adjuvant may be added tothe vaccine composition of the invention to boost the immune response toproduce more antibodies and longer-lasting immunity, thus minimizing thedose of antigen needed. Adjuvants may also be used to enhance theefficacy of a vaccine by helping to modify the immune response toparticular types of immune system cells: for example, by activating Tcells instead of antibody-secreting B cells depending on the purpose ofthe vaccine. Immunologic adjuvants are added to vaccines to stimulatethe immune system's response to the target antigen, but do not provideimmunity themselves. Examples of adjuvants include, but are not limitedto analgesic adjuvants; inorganic compounds such as alum, aluminumhydroxide, aluminum phosphate, calcium phosphate hydroxide; mineral oilsuch as paraffin oil; bacterial products such as killed bacteria(Bordetella pertussis, Mycobacterium bovis, toxoids); nonbacterialorganics such as squalene; delivery systems such as detergents (Quil A);plant saponins from Quillaja, soybean, or Polygala senega; cytokinessuch as IL-1, IL-2, IL-12; combination such as Freund's completeadjuvant, Freund's incomplete adjuvant; food-based oil such as Adjuvant65, which is based on peanut oil.

The contents of exosomes depends, in part, on the character of the cellsthat produce them. Cells can be genetically modified to configureexosomes produced by them. Fang et al., (PLOS, June 2007 vol.5:1267-1283) describe methods of engineering proteins to preferentiallytarget them toward exosomes. It was observed that (1) addition of bothmonoclonal mouse IgG to CD43 and polyclonal anti-mouse IgG antibodieswere sufficient to induce the sorting of CD43 to exosomes, (2) additionof a plasma membrane anchor was sufficient to target a protein toexosomes, (3) a synthetic cargo comprised of a plasma membrane anchorand two heterologous oligomerization domains (Acyl-LZ-DsRED) was sortedto exosomes, (4) highly oligomeric, plasma membrane-associatedretroviral Gag proteins (from EIAV, HTLV-1, RSV, MLV, MPMV, and HERV-K)were all sorted to ELDs and exosomes, and (5) a pair of heterologousoligomerization domains was necessary and sufficient to target HIV Gagto ELDs and exosomes. Elements, such as these, that traffic proteins toEVs, are referred to as “EV-trafficking elements.” Accordingly, anyprotein of interest can be modified in this way to traffic the proteintowards exosomes.

All patents and other publications; including literature references,issued patents, published patent applications, and co-pending patentapplications; cited throughout this application are expresslyincorporated herein by reference for the purpose of describing anddisclosing, for example, the methodologies described in suchpublications that might be used in connection with the technologydescribed herein. These publications are provided solely for theirdisclosure prior to the filing date of the present application. Nothingin this regard should be construed as an admission that the inventorsare not entitled to antedate such disclosure by virtue of priorinvention or for any other reason. All statements as to the date orrepresentation as to the contents of these documents is based on theinformation available to the applicants and does not constitute anyadmission as to the correctness of the dates or contents of thesedocuments.

The description of embodiments of the disclosure is not intended to beexhaustive or to limit the disclosure to the precise form disclosed.While specific embodiments of, and examples for, the disclosure aredescribed herein for illustrative purposes, various equivalentmodifications are possible within the scope of the disclosure, as thoseskilled in the relevant art will recognize.

For example, while method steps or functions are presented in a givenorder, alternative embodiments may perform functions in a differentorder, or functions may be performed substantially concurrently. Theteachings of the disclosure provided herein can be applied to otherprocedures or methods as appropriate. The various embodiments describedherein can be combined to provide further embodiments. Aspects of thedisclosure can be modified, if necessary, to employ the compositions,functions and concepts of the above references and application toprovide yet further embodiments of the disclosure. Moreover, due tobiological functional equivalency considerations, some changes can bemade in protein structure without affecting the biological or chemicalaction in kind or amount. These and other changes can be made to thedisclosure in light of the detailed description. All such modificationsare intended to be included within the scope of the appended claims.

Specific elements of any of the foregoing embodiments can be combined orsubstituted for elements in other embodiments. Furthermore, whileadvantages associated with certain embodiments of the disclosure havebeen described in the context of these embodiments, other embodimentsmay also exhibit such advantages, and not all embodiments neednecessarily exhibit such advantages to fall within the scope of thedisclosure.

The technology described herein is further illustrated by the followingexamples which in no way should be construed as being further limiting.

EXAMPLES

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the present invention asshown in the specific embodiments without departing from the spirit orscope of the present invention as broadly described. The presentembodiments are, therefore, to be considered in all respects asillustrative and not restrictive. In order that the present inventionmay be readily understood and put into practical effect, particularpreferred embodiments will now be described by way of the followingnon-limiting examples.

Plasmids and Viruses

Plasmids were maintained in DH10B cells, grown in ampicillin-containingLB media, and purified from bacterial lysates using mini-prep andmidi-prep plasmid isolation kits (Promega). Automated Sanger DNAsequencing was performed using custom primers and dye-linkeddideoxynucleotides (Applied Biosystems). DNA sequence data wereassembled, maintained and analyzed using SnapGene software. The3×NLS-tdTomato, CD81mNG, BleoR, PuroR, HygR, BsdR, NeoR, and SB100Xcoding regions were codon-optimized for expression in human cells,synthesized in vitro, cloned into mammalian cell expression vectors, andsequence-confirmed prior to use. The ITR-left and ITR-right backbone ofthe Sleeping Beauty vectors(18,19) and the cis-acting elements of athird generation, replication-defective and self-inactivating lentiviralbackbone(57) were also synthesized in vitro, cloned into minimalbacterial plasmids, and sequence-confirmed prior to use. The EBNA1 andOriP sequences(22) were assembled by a combination of in vitro genesynthesis and excision from pCEP4 (ThermoFisher), cloned into a minimalplasmid vector, with sequence confirmation of all newly synthesizedsegments of DNA. Transgenes and polylinkers were inserted into transgenedelivery vectors using standard recombinant DNA cloning techniques.

TABLE 1 List of plasmids used in this study Plasmid Plasmid name codeVector type Enhancer/Promoter pcDNA3-3xNLS-tdTomato*-2a-BsdR* pJM825non-replicating plasmid CMV pCJM-3xNLS-tdTomato*-2a-NeoR* pJM1074non-replicating plasmid CMV pCJM-3xNLS-tdTomato*-2a-BsdR* pJM908non-replicating plasmid CMV pCJM-3xNLS-tdTomato*-2a-HygR* pJM912non-replicating plasmid CMV pCJM-3xNLS-tdTomato*-2a-PuroR* pJM916non-replicating plasmid CMV pCJM-3xNLS-tdTomato*-2a-bleoR* pJM904non-replicating plasmid CMV pC-CD81mNG*-2a-NeoR* pCG18 non-replicatingplasmid CMV pC-CD81mNG*-2a-BsdR* pCG14 non-replicating plasmid CMVpC-CD81mNG*-2a-HygR* pCG16 non-replicating plasmid CMVpC-CD81mNG*-2a-PuroR* pCG10 non-replicating plasmid CMVpC-CD81mNG*-2a-BleoR* pCG12 non-replicating plasmid CMVpS-CD81mNG*-2a-PuroR* pJM1277 non-replicating plasmid CMVpITRSB-C-CD81mNG*-2a-PuroR* pJM1358 Sleeping Beauty transposon CMVpITRSB-S-CD81mNG*-2a-PuroR* pJM1366 Sleeping Beauty transposon CMVpREP-C-CD81mNG*-2a-PuroR* pCG43 replicating episome CMVpREP-S-CD81mNG*-2a-PuroR* pJM1367 replicating episome CMVpLenti-C-CD81mNG*-2a-PuroR* pJM1291 lentiviral provirus CMVpLenti-S-CD81mNG*-2a-PuroR* pJM1293 lentiviral provirus CMV

TABLE 2 Vector series for transgene expression Plasmid Plasmid name codeVector type Enhancer/Promoter pC-2a-BleoR* pJM1245 non-replicatingplasmid CMV pC-2a-PuroR* pJM1242 non-replicating plasmid CMV pC-2a-HygR*pJM1247 non-replicating plasmid CMV pC-2a-BsdR* pJM1246 non-replicatingplasmid CMV pC-2a-NeoR* pJM1248 non-replicating plasmid CMV pC-pIpJM1329 non-replicating plasmid CMV pS-3a-BleoR* pJM1345 non-replicatingplasmid SFFV LTR pS-2a-PuroR* pJM1344 non-replicating plasmid SFFV LTRpS-2a-HygR* pJM1346 non-replicating plasmid SFFV LTR pS-2a-BsdR* pJM1400non-replicating plasmid SFFV LTR pS-2a-NeoR* pJM1347 non-replicatingplasmid SFFV LTR pS-pI pJM1330 non-replicating plasmid SFFV LTRpITRSB-C-2a-BleoR* pJM1384 Sleeping Beauty transposon CMVpITRSB-C-2a-PuroR* pJM1355 Sleeping Beauty transposon CMV pITRSB-C2a-HygR* pJM1385 Sleeping Beauty transposon CMV pITRSB-C-2a-BsdR*pJM1401 Sleeping Beauty transposon CMV pITRSB-C-2a-NeoR* pJM1386Sleeping Beauty transposon CMV pITRSB-C-pI pJM1356 Sleeping Beautytransposon CMV pITRSB-S-2a-BleoR* pJM1389 Sleeping Beauty transposonSFFV LTR pITRSB-S-2a-PuroR* pJM1388 Sleeping Beauty transposon SFFV LTRpITRSB-S-2a-HygR* pJM1390 Sleeping Beauty transposon SFFV LTRpITRSB-S-2a-BsdR* pJM1402 Sleeping Beauty transposon SFFV LTRpITRSB-S-2a-NeoR* pJM1391 Sleeping Beauty transposon SFFV LTRpITRSB-S-pI pJM1393 Sleeping Beauty transposon SFFV LTRpLenti-C-2a-BleoR* pJM1350 Lentiviral provirus CMV pLenti-C-2a-PuroR*pJM1349 Lentiviral provirus CMV pLenti-C 2a-HygR* pJM1351 Lentiviralprovirus CMV pLenti-C-2a-BsdR* pJM1377 Lentiviral provirus CMVpLenti-C-2a-NeoR* pJM1352 Lentiviral provirus CMV pLenti-C-pI pJM1354Lentiviral provirus CMV pLenti-S-2a-BleoR* pJM1360 Lentiviral provirusSFFV LTR pLenti-S-2a-PuroR* pJM1359 Lentiviral provirus SFFV LTRpLenti-S-2a-HygR* pJM1361 Lentiviral provirus SFFV LTR pLenti-S-2a-BsdR*pJM1378 Lentiviral provirus SFFV LTR pLenti-S-2a-NeoR* pJM1362Lentiviral provirus SFFV LTR pLenti-S-pI pJM1364 Lentiviral provirusSFFV LTR pREP-C-2a-BleoR* pJM1403 Lentiviral provirus CMVpREP-C-2a-PuroR* pJM1403 Lentiviral provirus CMV pREP-C 2a-HygR* pJM1404Lentiviral provirus CMV pREP-C-2a-BsdR* pJM1405 Lentiviral provirus CMVpREP-C-2a-NeoR* pJM1406 Lentiviral provirus CMV pREP-C-pI pJM1335Lentiviral provirus CMV

To make replicating-defective, self-inactivating lentiviruses, 293Tcells were transfected with a mixture of 4 plasmids: the lentiviralvector, a Gag-Pol expression vector, a Rev expression vector, and aVSV-G expression vector(57). The transfected cells were incubated for 3days. The tissue culture supernatant was collected, spun at 5000×g for15 minutes to remove cells and cell debris, and the resultingsupernatant was passed through a 0.45 μm filter to generate anunconcentrated virus stock.

Cell Culture, Transfection, Transduction, and Antibiotic Selection

All cell lines were grown in a tissue culture incubator maintained at 5%CO₂, 90-99% humidity, and 37° C. HEK293 and 293T cells were obtainedfrom the American Type Culture Collection (ATCC) and grown in DMEM (highglucose; Gibco/BRL) supplemented with 10% fetal calf serum (FCS;ThermoFisher). COST cells were also grown in DMEM (high glucose;Gibco/BRL) supplemented with 10% fetal calf serum (FCS; ThermoFisher).293F cells (ThermoFisher) were grown either as suspension cells inchemically-defined Freestyle media (ThermoFisher) in uncoated tissueculture shaker flasks on a shaking platform at 110 rpm, or in DMEMsupplemented with 10% FCS in standard, coated tissue culture flasks ordishes. Cells were transfected using Lipofectamine 2000 reagent(ThermoFisher). In brief, 5 μgs of plasmid DNA was diluted into 0.3 mlsof Opti-Mem medium (Gibco/BRL), while 15 μls of Lipofectamine 2000 wasdiluted into a separate 0.3 mls of Opti-Mem medium, and both mixtureswere incubated separately for 5 minutes. The two mixtures were mixedtogether and incubated for a further 15 minutes. Next, growth medium wasremoved from a T-25 coated tissue culture flask containing HEK293 cellsat ˜70-90% confluency. The cells were then washed with 5 mls of Opti-Memmedium (at 37° C.), all liquid was removed, and the 0.6 ml mixture ofDNA, Lipofectamine 2000 and Opti-Mem was added to the flask. Aftergentle rocking to distribute the mixture across the entire flasksurface, the flask was incubated for 15-20 minutes in a tissue cultureincubator. The DNA/Lipofectamine 2000/Opti-MEM solution was thenremoved, 5 mls of DMEM+10% FCS was added to each flask, and the cellswere then returned to the incubator for between 1 and 2 days, dependingon the experiment. For lentiviral transductions, 1 ml of unconcentratedvirus stock was added to 10 mls of culture media in the presence of 6μg/ml polybrene and ˜3×10⁶ cells in a 10 cm tissue culture dish,incubated for 2 days, and then washed.

Antibiotic-resistant cell lines were generated from transfected ortransduced cell HEK293

cells by splitting cells onto 150 mm dishes containing DMEM, 10% FCS,and the appropriate antibiotic. Antibiotics were used at the followingconcentrations: 400 μg/ml G418, 20 μg/ml blasticidin, 400 μg/mlhygromycin B, 3 μg/ml puromycin, and 200 μg/ml zeocin (theseconcentrations approximately twice that required to kill HEK293 cells(data not shown)). Transfected cell populations were re-fed every 3-4days until distinct, drug-resistant clones were large enough to be seenby eye and all antibiotic-sensitive cells between the drug-resistantcolonies had died off, typically 10-14 days. The drug-resistant cellsfrom each transfected population were then pooled to create polyclonalcell lines and expanded until they had grown for 4 weeks from the dateof transfection in selective media. Each cell line was then processedfor flow cytometry, fluorescence microscopy, and/or immunoblot.

Flow Cytometry

Cells were suspended by trypsinization, washed in Hank's buffered salinesolution (HBSS) and resuspended at a concentration of 1×10⁷ cells per mlin cold (4° C.) HBSS containing 0.1% FBS. Cell suspensions weremaintained on ice, diluted to a concentration of 1×10⁶ cells per ml, andexamined for tdTomato or mNeonGreen fluorescence by flow cytometry on aBeckman MoFlo Cell Sorter equipped with 355 nm, 488 nm, and 633 nmlasers set to the appropriate detection wavelength. The relativebrightness was determined for thousands of individual cells in each cellline using Beckman MoFlo software and reported as scatter plots, averagerelative brightness, and coefficient of variation.

Immunofluorescence

Cells were seeded onto sterile (autoclaved) borosilicate cover glassesin tissue culture dishes and grown overnight in normal media. The coverglasses were removed from the tissue culture dishes, washed, and fixedin 3.7% formaldehyde in Dulbecco's modified phosphate-buffered saline(DPBS), pH 7.4, for 15 minutes. The cover glasses were then washed inDPBS, incubated in DPBS containing DAPI (5 μg/ml) for 15 minutes, washed5 times in DPBS, and mounted on a glass slide containing ˜8 μls ofmounting solution (90% glycerol, 100 mM Tris pH8.5, 0.01%para-phenylenediamine). After removal of excess mounting solution, thecells were examined using a Nikon Eclipse TE200 microscope equipped withNikon S Fluor 20x, 0.75 aperture objective and an Andor Neo sCMOSDC-152Q-COOF digital camera. Images were processed using Photoshop andassembled in illustrator (Adobe).

Immunoblot

Equal numbers of each HEK293-derived cell line were lysed in SDS-PAGEsample buffer (20% glycerol, 4% SDS, 120 mM Tris-HCl (pH 6.5), 0.02%bromophenol blue) at room temperature, then frozen and thawed once. Thethawed samples were then boiled for 10 minutes, spun at 10,000×g for 2minutes to pellet insoluble materials, loaded onto 4-15% polyacrylamidegradient gels (Bio-Rad), and electrophoresed according to themanufacturer's suggestions. Proteins were then transferred toimmobilon-P membranes (Amersham), incubated in blocking solution (5%non-fat dry milk in TBST (138 mM NaCl, 2.7 mM KCl, 50 mM Tris, pH 8.0,0.05% Tween-20) for two hours, and then in blocking solution containingprimary antibodies overnight at 4° C. (rabbit polyclonal anti-p2aantibody was used at a dilution of 1:1000 and anti-actin antibodies wereused at a dilution of 1:1000). The membranes were washed 5 times in TBSTand then incubated with blocking solution containing secondaryantibodies conjugated with horseradish peroxidase (HRP) at a dilution of1:5000 for 1 hour. The membranes were washed 5 times with TBST,incubated in HRP-activated chemiluminescence detection solution(Amersham ECL Western Blotting Detection Reagents; cat #RPN2106), andimaged using a GE Amersham Imager 600. Images were exported as JPEGfiles, analyzed using ImageJ software, and processed using Photoshopsoftware (Adobe).

Exosome Analysis

293F-derived cells were grown in sterile shaker flasks containing 80 mlsof Freestyle media for 5 days at a starting concentration of 5×10⁵cells/ml. Clarified tissue culture supernatants (CTCSs) of each culturewere then generated by centrifuging the cultures at 5000×g for 15minutes to remove all cells, and passing the resulting supernatantthrough a 0.22 μm filter to remove large cell debris. ˜80 mls of theCTCS was then concentrated to ˜0.5 ml using a 100 kDa molecular weightcut-off angular filtration unit (Centricon-70) according to themanufacturer's suggestions. The resulting samples were then passed overan Izon qEV 35 nm size exclusion chromatography column using PBS, pH 7.4as column buffer and collecting 0.5 mls fractions. Fractions 4, 5, and 6contained exosomes (data not shown) and were pooled to generate eachexosome preparation. These were examined by nanoparticle trackinganalysis using a Particle Matrix ZetaView Twin 488 & 640(PMX-220-12C-R4) according to the manufacturer's suggestions.

Heterogeneous Expression from NeoR and BsdR-Linked Transgenes

The classic approach to generating a transgenic mammalian cell line isto transfect (or transduce) the cells with a plasmid (or virus) carryingtwo genes, one encoding the recombinant protein of interest, and theother encoding a dominant selectable marker that confers resistance toan otherwise toxic antibiotic(4,5). The NeoR gene confers resistance tothe protein synthesis inhibitor G418 (geneticin) and is the selectablemarker on many if not most mammalian cell expression vectors, includingpcDNA3. To document the outcomes of a classic, two-gene transgenesisexperiment using the NeoR marker gene, the present inventors firstcreated a derivative of pcDNA3 designed to encode 3×NLS-tdTomato, a formof the red fluorescent protein tdTomato(45) that carries three copies ofa nuclear localization signal (NLS)(46) at its N-terminus (FIG. 1A).However, the present inventors also wanted to assess the expression of3×NLS-tdTomato after the selection for a more directly linked selectablemarker. The present inventors therefore expressed 3×NLS-tdTomato from abicistronic ORF encoding the porcine teschovirus 2a peptide(47) and theblasticidin deaminase enzyme(29). As a result, it should be possible touse this one plasmid to compare recombinant protein expression profilein transgenic cell lines generated by either a two-gene selectionstrategy or a bicistronic, stoichiometrically balanced selectionstrategy.

HEK293 cells were transfected with this plasmid (pJM825), grown for oneday in normal media, followed by transferring half of the transfectedcells into G418-containing media and half into blasticidin-containingmedia. Thousands of antibiotic-resistant clones emerged from eachselection. After two weeks, these clones were pooled to create twomixed-clone cell lines, which were then grown for an additional twoweeks in antibiotic-containing media and assayed for 3×NLS-tdTomatoexpression by flow cytometry (FIGS. 1B,C). After assaying thousands ofcells from each line, it was observed that ˜50% of cells in theG418-resistant line cells lacked detectable levels of 3×NLS-tdTomatoexpression. This is not surprising, given that the 3×NLS-tdTomato generepresents a sizeable proportion of the transfected plasmid, and willtherefore be disrupted in a significant proportion of G418-resistantcell lines due to the random nature of plasmid linearization that occursduring transgene insertion into host chromosomes(4,5). The moretroubling observation was that the levels of 3×NLS-tdTomato fluorescenceamong the expressing cells varied so widely, as if resistance to G418had little if any correlation with transgene expression levels. Flowcytometric analysis of the blasticidin-resistant cell line revealed afar lower percentage of non-expressing cells, consistent with the factthat each blasticidin deaminase enzyme is synthesized only after thesynthesis of one 3×NLS-tdTomato protein. Unfortunately, this cell linealso displayed a pronounced heterogeneity in 3×NLStdTomato fluorescence,indicating that resistance to BsdR also had little if any correlationwith transgene expression levels. Consistent with these conclusions, thepresent inventors observed a pronounced heterogeneity in 3×NLS-tdTomatoexpression when they stained these cell lines with the DNA stain DAPIand examined them by fluorescence microscopy (FIGS. 1D-I).

Choice of Selectable Marker Affects Recombinant Protein Expression

These results raised the question of whether all dominant selectablemarkers yield cell lines with similarly low and heterogeneous levels ofrecombinant protein expression. To explore this issue, the presentinventors created a new set of bicistronic expression vectors in which3×NLS-tdTomato expression was linked via the p2a peptide to the NeoR(3),BsdR(29), HygR(27), PuroR(26), and BleoR(28) markers (FIG. 2A). HEK293cells were transfected with each of these plasmids, grown for a day innormal media, and then incubated for two weeks in media containing G418,blasticidin, hygromycin, puromycin, or zeocin, respectively. Clones fromeach transfection were then pooled to generate five distinct cell lines,which were then examined for 3×NLS-tdTomato expression by flow cytometry(FIGS. 2B-G; Table 3).

TABLE 3 Flow cytometry data for cell lines expressing 3xNLS-tdTomatofrom non-replicating plasmids Average relative % Non-expressing Cellline brightness; c.v. cells HEK293/pCJM-3xHLS-tdTomato*-2a-NeoR* 458;103  22% HEK293/pCJM-3xNLS-tsTomato*-2a-BsdR* 522; 82   3%HEK293/pCJM-3xNLS-tdTomato*-2a-HygR* 794; 62 0.40%HEK293/pCJM-3xNLS-tdTomato*-2a-PuroR* 803; 44 0.30%HEK293/pCJM-3xNLS-tdTomato*-2a-BleoR* 1754; 46 0.20% HEK293 3; 153  100%*Codon optimized

The NeoR- and BsdR-resistant cell lines displayed the lowest averagerelative brightness and high degrees of cell-to-cell variation in3×NLS-tdTomato fluorescence (458, with a coefficient of variance(c.v.)=103; and 522, with c.v.=82, respectively). In contrast, the HygR-and PuroR-based cell lines displayed higher and more homogeneous levelsof 3×NLS-tdTomato expression (794, c.v.=62; and 803, c.v.=44,respectively), and the BleoR-based cell line displayed the highest andmost homogeneous expression of 3×NLS-tdTomato (1754, c.v.=46).

Choice of Selectable Marker Affects Exosomal Protein Expression

To determine whether these effects were specific to 3×NLS-tdTomato orcould be extrapolated to other recombinant proteins, the presentinventors created another set of vectors designed to express an exosomalcargo protein, CD81(48), an integral plasma membrane protein that ishighly enriched in exosomes(49). Moreover, the present inventorsexpressed CD81 as a fusion protein with the green fluorescent proteinmNeonGreen(50), allowing the detection of this protein (CD81mNG) byfluorescence-based techniques (FIG. 3A). HEK293 cells were transfectedwith these five plasmids and the antibiotic-resistant clones were pooledto create five polyclonal cell lines. These lines were then examined byflow cytometry to measure the relative levels of CD81mNG fluorescence inthousands of cells within each population (FIGS. 3B-G). The NeoR- andBsdR-derived cell lines once again displayed the lowest and mostheterogeneous expression of their linked recombinant protein, withrelative CD81mNG brightness levels of 465 (c.v.=93) and 316 (c.v.=126),respectively. In contrast, the cell lines derived by transfection withthe HygR- and PuroR-based plasmids displayed higher and more homogeneouslevels of expression (average relative CD81mNG brightness of 790(c.v.=63) and 1000 (c.v.=63), respectively. Once again, the cellsderived by transfection with the BleoR-based plasmid displayed thehighest and most homogeneous levels of transgene expression (1749;c.v.=55). Similar results were observed when these cell lines wereinterrogated by fluorescence microscopy (FIG. 4 ) or by immunoblotanalysis (FIG. 5 ), the latter of which showed an ˜10-fold increase inthe expression of CD81mNG in the BleoR-derived cell line relative to theBsdR-derived or NeoR-derived cell lines, with intermediate levels ofexpression in the HygR- and PuroR-derived cell lines.

Taken together, the preceding results indicate that each selectablemarker and antibiotic establish a distinct threshold of transgeneexpression below which no cell can survive, with the most permissivemarkers (NeoR and BsdR) allowing survival at low, medium or high levelsof transgene expression, the most restrictive marker (BleoR) killing allbut the most highly expressing transgenic cells, and cells selectedusing the HygR or PuroR markers falling somewhere in between. If thishypothesis is correct, similar results should be observed in orthogonalmeasurements of CD81mNG expression. The present inventors thereforeprocessed these five cell lines for fluorescence microscopy, stainingeach sample with DAPI to stain the cell nuclei. The resulting imagesshow that CD81mNG expression was lowest in the G418-resistant andblasticidin-resistant cell lines, higher in the hygromycin-resistant andpuromycin-resistant cell lines, and highest in the zeocin-resistant cellline (FIG. 4 ). These conclusions were also reinforced by immunoblotanalysis of cell lysates processed using antibodies specific for (a) the2a peptide appended to the C-terminus of CD81mNG proteins expressed fromthese vectors and (b) the cytoplasmic protein actin, which serves as aloading control (FIG. 5 ). These immunoblot data were quantified and thestatistical analysis of the data revealed CD81mNG expression was roughly10-fold higher in the BleoR-derived cell line than either theBsdR-derived or NeoR-derived cell lines, with intermediate levels ofexpression in the HygR and PuroR-derived cell lines.

Genetic Engineering of Exosomes

CD81 is among the most highly enriched exosomal proteins known(49) andhas high potential as a carrier molecule for modifying exosome content.To determine whether its incorporation into exosomes is impacted by itslevel of expression in the exosome-producing cells, the presentinventors transfected 293F cells (a derivative of HEK293 cells) with thePuroR- and BleoR-linked CD81mNG expression vectors described above(pC-CD81mNG*-2a-PuroR* and pC-CD81mNG*-2a-BleoR*). A day later,selection for antibiotic-resistant clones was initiated, which wereexpanded as pools of puromycin-resistant and zeocin-resistant cells.These two cell lines were then inoculated into chemically-defined mediain shaker flasks and grown for 5 days. The cells were then removed fromthe conditioned media and exosomes were purified by a combination of lowspeed centrifugation, size exclusion filtration, filtration-basedconcentration, and size exclusion chromatography. The two exosomepreparations were then interrogated by nanoparticle tracking analysis(NTA) using a Particle Metrix Zetaview PMX220(51) to measure theconcentrations, sizes, and CD81mNG fluorescence of exosomes in eachsample (FIG. 6 ; Table 4).

TABLE 4 Exosome sizes, concentration, and fluorescence data asdetermined by NTA exosome-producing cell line number of exosomes averagesize 293F/pC-CD81mNG*-2a-PuroR* (total) 3.8 × 10{circumflex over( )}11     112 nm 293F/pC-CD81mNG*-2a-PuroR* (fluorescent) 9.9 ×10{circumflex over ( )}10 (26%) 109 nm 293F/pC-CD81mNG*-2a-BleoR*(total) 1.4 × 10{circumflex over ( )}12     114 nm293F/pC-CD81mNG*-2a-BleoR* (fluorescent) 9.8 × 10{circumflex over ( )}11(70%)  97 nm

Both preparations contained large numbers of extracellular vesicles, thevast majority of which had the size expected of exosomes (mean diametersof 112 nm and 114 nm, respectively), confirming that exosomes werepurified, and not the larger microvesicle class of extracellularvesicles. The puromycin-resistant cell line, which expresses high levelsof CD81mNG, released a population of exosomes in which ˜25% displayeddetectable levels of CD81mNG fluorescence. In contrast, thezeocin-resistant cell line, which expresses higher levels of CD81mNG,released a population of exosomes in which 70% carried detectable levelsof CD81mNG, a >2-fold increase in exosome occupancy that is consistentwith a stochastic model of exosome biogenesis (42).

Consistent Transgene Expression Across Different Platforms

The present inventors predicted that the most restrictive selectablemarkers such as BleoR and PuroR establish a high threshold of transgeneexpression. If this prediction is correct, then these markers should atleast partly blunt the impact of other vector design variables on thelevels of recombinant protein expression in polyclonal pools ofantibiotic-resistant cells. To explore this possibility, the presentinventors created a series of eight DNA vectors that express theidentical recombinant protein (CD81mNG-2a-PuroR) from two differenttranscriptional control elements (CMV(9) or SFFV long terminal repeat(LTR)(8)) delivered by four distinct vector systems: non-replicatingplasmids (pC and pS), Sleeping Beauty transposons (pITRSB-C andpITRSB-S)(18,19), Epstein Barr Virus (EBV)-based episomes (pREP-C orpREP-S)(22), or replication-defective, self-inactivating lentiviruses(Lenti-C or Lenti-S) (FIGS. 7A, B). HEK293 cells were transfected withthe six naked DNA vectors and transduced with the two lentiviralvectors, followed by selection of puromycin-resistant clones to createeight polyclonal cell lines. These were examined by flow cytometry(FIGS. 7C-J; Table 5), revealing that all eight cell lines displayedroughly similar CD81mNG fluorescence profiles.

TABLE 5 Flow cytometry data for puromycin-resistant HEK293 cellstransfected or transduced with different vector systems average relative% non-expressing cell line brightness; c.v. cellsHEK293/pC-CD81mNG*-2a-PuroR* 202; 55 >0.01% HEK293/plTRSB-C-CD81mNG*-2a-PuroR* 294; 60 >0.01% HEK293/pREP-C-CD81mNG*-2a-PuroR* 364; 68 0.04%HEK293/pLenti-C-CD81mNG*-2a-PuroR* 212; 29 0.01%HEK293/pS-CD81mNG*-2a-PuroR* 216; 60 0.40%HEK293/plTRSB-S-CD81mNG*-2a-PuroR* 355; 62   0%HEK293/pREP-S-CD81mNG*-2a-PuroR* 253; 96 0.03%HEK293/pLenti-S-CD81mNG*-2a-PuroR* 273; 41 >0.01% 

To test whether similar results would be observed in the absence ofpuromycin selection, the present inventors transfected HEK293 cells withthe same six DNA vectors at two days post-transfection (FIGS. 7K-P;Table 6), a time when unselected transgene expression is highest.

TABLE 6 Flow cytometry data for transiently transfected HEK293 cellpopulations average relative % non-expressing cell line brightness; c.v.cells HEK293/pC-CD81mNG*-2a-PuroR* 86; 355 37%HEK293/plTRSB-C-CD81mNG*-2a-PuroR* 37; 453 59%HEK293/pREP-C-CD81mNG*-2a-PuroR* 330; 253 3.8% HEK293/pS-CD81mNG*-2a-PuroR* 37; 453 59%HEK293/plTRSB-S-CD81mNG*-2a-PuroR* 9.3; 459 81%HEK293/pREP-S-CD81mNG*-2a-PuroR* 20; 366 68%

These transiently-transfected cell populations contained many morenon-expressing cells, a much lower average level of CD81mNGfluorescence, and much higher cell-to-cell variation of CD81mNGexpression. These results indicate that the CMV-based vectors drovehigher CD81mNG expression than the SFFV LTR-based vectors, especiallywhen delivered on an EBV-based replicating vector. These results supportthe idea that the similarity in CD81mNG in expression seen in the eightpuromycin-selected cells is due to the restrictive nature of puromycinselection following transfection with PuroR-linked transgenes. Thepresent inventors also collected data on the time course of unselected,CMV-driven, CD81mNG expression, which confirmed that transgeneexpression declines rapidly in the absence of antibiotic selection frommultiple gene delivery platforms.

Parallel Results in Monkey COS7 Cells

To determine whether choice of selectable marker has a similar effect ontransgene expression in other mammalian cell lines, the presentinventors transfected the simian virus 40-transformed African greenmonkey kidney cell line COS-7 with the non-replicating plasmids designedto express CD81mNG from polycistronic ORFs linked by a 2a peptide to theNeoR(3) BsdR(29), HygR(27), PuroR(26), and BleoR(28) markers (FIG. 3A).Polyclonal cell lines were generated from each population of transfectedcells by culturing them in their cognate antibiotic for 10-14 days,pooling clones from each transfection, and then expanding them underselection for another 1-2 weeks. These cell lines were then stained andexamined by fluorescence microscopy, which revealed the same pattern oflinked CD81mNG expression: highest in the cell lines selected in zeocin,lowest in cell lines selected with blasticidin or G418, and intermediatein cell lines selected with hygromycin or puromycin (FIG. 8 ).

Discussion—Part 1

The creation of transgenic mammalian cells is a critical step in manybiomedical research projects. However, there is no simple, inexpensive,and rapid method for generating transgenic cell lines that express highand relatively homogeneous levels of linked recombinant proteins. Thepresent inventors explored herein the impact that the choice ofselectable marker has on the levels of a linked recombinant protein, andfound that it can have up to a 10-fold effect on expression level.Moreover, the present inventors established that the choice ofselectable marker also has a pronounced effect on the cell-to-cellvariation in transgene expression, with the highest variationcorrelating with the lowest average expression and the lowestcell-to-cell variation observed in the highest-expressing polyclonalcell lines.

The simplest interpretation of these observations is that eachselectable marker-antibiotic pair establishes a threshold of transgeneexpression below which no cell can survive. The present inventorsanticipate that these thresholds are determined, at least in part, byeach marker protein's mechanism of action, intrinsic activity, andstability within the cell. Given that these variables are likely to bedistinct for nearly all proteins, it is not surprising that eachmarker/antibiotic pair established a distinct threshold of transgeneexpression. More specifically, this model predicts that highly efficientand long-lived selectable marker proteins inactivate their cognateantibiotic even at very low levels of expression. As a result, their useresults in the survival of cells that express almost any level of thelinked recombinant protein, which manifests in pooled, polyclonal celllines as a low average level of transgene expression and a high degreeof cell-to-cell variation in transgene expression levels. Theseproperties correspond relatively well to those observed for cell linesgenerated using the NeoR or BsdR markers, indicating that the NeoR andBsdR selectable marker enzymes may be highly active, stable, or both. Asfor the higher levels of transgene expression in hygromycin-resistant orpuromycin-resistant cell lines, the present inventors posit that theHygR and PuroR enzymes may be less active, less stable, or both. Andfinally, the fact that the BleoR marker consistently yielded cell lineswith the highest and least heterogeneous levels of transgene expressionindicates that it has the lowest activity of all selectable markerproteins, consistent with its non-catalytic mechanism of zeocininactivation. This interpretation of our results is actually consistentwith the known mechanism of BleoR-mediated resistance to zeocin, whichis non-catalytic and involves its chelation of zeocin at a 1:1 molarratio(52).

A major outcome of this study is the realization that the BleoR markercan be used to quickly create polyclonal cell lines expressingrelatively high and homogeneous levels of a linked recombinant protein.Although the data presented herein establishes this for only twoproteins, follow-on studies indicate that it works for many otherproteins (e.g. EGFR, PD-L1, HEMO, etc.). While the levels of expressionattained by this system may still be less than those achieved by morelabor-intensive approaches, the present approach to recombinant proteinexpression is significantly faster and less time consuming, and clearlysuperior to the traditional, two-gene strategy for making transgenicmammalian cells. As for how to make best use of the expression systemdescribed herein, the present inventors contemplate transfecting a verylarge population of cells (>1×10⁶), in part because the zeocin selectionkills all but the most highly expressing transgenic clone, and in partbecause the number of initial zeocin-resistant clones determines thetime required to generate a working cell line. That being said, use ofthe BleoR marker and zeocin is not free of concern, as zeocin killscells by binding DNA and inducing DNA damage(54). In fact,zeocin-induced DNA damage can even occur in BleoR-expressing,zeocin-resistant cell lines(55). While the PuroR-derived cell linesdisplayed lower levels of transgene expression, the present inventorsare unaware of long-term damage associated with extended growth ofPuroR-derived cell lines in its cognate antibiotic, and the same can besaid for HygR/hygromycin-based selection of transgenic cell lines.

These findings are also relevant to the creation of clonal transgeniccell lines. After all, they represent strong evidence that use of theBleoR marker eliminates cells that express low or medium levels of alinked recombinant protein, with similar but lesser effects attained byuse of the PuroR or HygR markers. Similar considerations recommend useof the BsdR marker for projects that require a low level of transgeneexpression, as the cell lines generated using the BsdR marker andblasticidin selection allowed survival of poorly-expressing cell clones.As for the NeoR marker, the present inventors cannot recommend its usefor any purpose, largely because the NeoR-derived cell lines displayedthe greatest variation in transgene expression, contained the highestpercentage of non-expressing cells, and exhibited time-dependent changesin recombinant protein expression. Taken together, these considerationsraise the possibility that the NeoR gene, which has been used more oftenthan any other selectable marker (see addgene.org), may in fact be theleast useful of all.

These studies were performed primarily in the context of expressingCD81, an exosomal protein. The present inventors did this because theywanted to know whether their findings related to transgene expressionmight inform their approach to exosome engineering. A variety ofobservations support the hypothesis that exosome biogenesis isessentially a stochastic process in which the content of any individualexosome is determined by the local concentrations of exosome cargomolecules in the vicinity of a nascent vesicle budding event(42). Underthis model, increasing the expression of an exosomal cargo proteinwithin the cell should lead to its presence on a higher percentage ofexosomes. Consistent with this prediction, the present inventorsobserved that 293F cells expressing CD81mNG from a PuroR-linkedtransgene, which selects for high transgene expression, released apopulation of exosomes in which ˜25% of the vesicles containedmNeonGreen fluorescence. In contrast, 293F cells expressing CD81mNG froma BleoR-linked transgene, which selects for significantly highertransgene expression, released a population of exosomes in which ˜70%contained mNeonGreen fluorescence, an ˜2.5-fold increase in exosomeoccupancy by this engineered cargo molecules. Taken together, theseobservations indicate that the choice of selectable marker is animportant consideration in the genetic modification of exosome content,and is therefore relevant to the production of exosome-based therapies,standards and controls.

The general relevance of the present findings can only be determined bytesting this system in the context of other recombinant proteins, othercell lines, and other research environments. To facilitate this processthe present inventors have created a suite of vectors that are designedto drive the bicistronic expression of recombinant proteins in framewith (a) thep2a peptide and (b) the NeoR, BsdR, HygR, PuroR, or BleoRcoding sequences, transcribed from either the CMV enhancer/promoter orthe SFFV LTR, and carried on simple plasmids, Sleeping Beautytransposons, EBV-based episomes, or lentiviral vectors (FIGS. 9 and 10). While one cannot predict exactly which vector will yield the desiredtransgene expression characteristics for any given experiment, thesimplest interpretation of the present results is that high andhomogeneous expression of recombinant proteins may be easiest to achieveby delivering the transgene via a Sleeping Beauty transposon, using theSFFV LTR to drive its transcription, and by linking its expression tothe BleoR marker.

Degron Tagging of Selectable Markers

The presented inventors generated Sleeping Beauty transposons in whichthe CMV promoter was used to drive the expression of bicistronic openreading frames (ORFs) encoding (i) the fluorescent protein mCherry, (ii)an 18 amino acid-long viral 2a peptide (p2a), and (iii) an array ofdifferent selectable marker genes. These marker genes consisted ofpreviously characterized, codon-optimized genes encoding the BleoR,BsdR, NeoR, HygR, and PuroR proteins, as well as forms of each that weretagged at their N-terminus with the destabilization domains (DD) fromhuman estrogen receptor (ER50) and Escherichia coli dihydrofolatereductase (ecDHFR). (FIG. 11 ). These 15 vectors were then transfectedinto 293F cells. Shortly after transfection, expression of the SleepingBeauty transposase protein (SB100X) from the flanking region of theplasmid results in mobilization of the plasmid-borne transposon from thetransfected plasmid DNA into the host cell genome. Two days later, thecells were placed into complete growth media containing the cognateantibiotic and fed every 2-5 days for 10-15 days, resulting in death ofnon-transgenic cells and selection of numerous antibiotic-resistant cellclones. At this point, the clones generated from each transfectionpooled, expanded, and interrogated for mCherry expression by flowcytometry.

An Improved BleoR Marker

Transfection of HEK293 cells with vectors carrying the BleoR marker,followed by selection in zeocin-containing media, generates cell lineswith the highest average levels of linked recombinant proteinexpression. It should be noted that BleoR-based vectors also generatethe lowest percentage of antibiotic-resistant clones, for the very samereason, which is that only those transgenic cell lines with the veryhighest levels of transgene expression are able to survive. When thepresent inventors transfected cells with vectors carrying degron-taggedforms of the BleoR gene, the number of clones was even lower for theER50BleoR and none were obtained for cell transfected with the vectorcarrying the ecDHFRBleoR marker. As for the BleoR-selected andER50BleoR-selected cell lines that were generated following selection,flow cytometry revealed that degron tagging the BleoR marker resulted inan ˜2.5-fold increase in transgene expression. Specifically, it wasobserved that the mean mCherry fluorescence brightness increased from16,024 (arbitrary units) in the BleoR cell line to 37,141 in theER50BleoR cell line (FIG. 12 ). Given that the BleoR marker was alreadyknown to drive the highest and most homogeneous levels of linkedrecombinant protein expression, this represents a 2.5-fold increase tothe upper limit of selectable marker performance, at least for dominantselectable markers.

Improved Blasticidin-Resistance Genes

In the above analysis of the effect of selectable marker choice ontransgene expression levels, the present inventors demonstrated that itcould have as much as a 10-fold effect on the expression levels of alinked recombinant protein. The present inventors show here that thiseffect can be even larger, as much as 28-fold, which was the differencein mean mCherry brightness that was observed for HEK293 cells generatedusing the BsdR marker as compared to those generated using the ER50BleoRmarker (1,308 vs 37,141; FIG. 12 ). As for whether the BsdR marker couldbe improved by degron tagging, the present inventors' observationsshowed that this approach to marker gene improvement led to 5-fold and6-fold higher levels of mCherry expression for cells generated using theER50BsdR and ecDHFRBsdR markers, respectively (FIG. 12 ). Importantly,this elevated transgene expression levels that were only ˜2-fold lowerthan those achieved using the BleoR marker, and ˜4.5-fold lower thanachieved with the ER50BleoR marker. These improvements are significantand raise the possibility that blasticidin, which has the most rapidcell-killing kinetic of the five antibiotics used in mammalian celltransgenesis experiments, can now be more widely used in mammalian celltransgenesis experiments.

Limited Effect of Degron-Tagging on the NeoR and HygR Genes

For most of the past 40 years, the NeoR gene was the most commonly useddominant selectable marker in mammalian cell transgenesis. However, thepresent inventors' study establishing that choice of selectable markerhad a pronounced effect on transgene expression levels revealed that theNeoR gene was one of the two most poorly-performing selectable markergenes, routinely yielding cell lines with the lowest average levels oftransgene expression and the highest degree of cell-to-cell variabilityin transgene expression. Degron tagging did not improve this marker bymuch, as the highest levels of linked recombinant protein expression,observed for cells selected with the ecDHFRNeoR marker, were at best 30%higher than in cells selected with the NeoR marker (FIG. 12 ).

Degron tagging alone had even less of an effect on HygR performancecharacteristics, with no improvement observed for ER50HygR and only 20%improvement observed for ecDHFRHygR (FIG. 12 ). However, in the courseof creating the ecDHFRHygR gene, the present inventors accidentallygenerated a C-terminal truncation mutant (ecDHFRHygR*) through aPCR-generated frameshift mutation. This selectable marker displayed an˜70% increase in linked recombinant protein expression, and highlightsthe potential for combining degron tagging with random mutagenesis togenerate improved selectable marker genes.

Degron Tagging of the PuroR Gene

Degron tagging was also applied to the PuroR gene, but it was far lesseffective than what was observed for the BleoR or BsdR genes.Specifically, flow cytometry experiments revealed that cell linesgenerated using the ER50 and ecDHFR destabilization domains expressed˜70% higher expression of the linked recombinant protein (FIG. 12 ).While the magnitude of these effects was less than hoped for, it shouldbe noted that the levels of transgene expression selected by theER50PuroR and ecDHFRPuroR markers were nevertheless higher than anyother selectable markers, save for the BleoR and ER50BleoR markers.

Identification and Characterization of the PuroR2 Gene

The dominant selectable markers that form the foundation of theaforementioned studies were all cloned decades ago at a time when theutility of a potential selectable marker gene was thought to beproportional to the numbers of antibiotic-resistant clones that wereobtained following a standard transfection and selection protocol. Thisproperty of yielding large numbers of antibiotic-resistant clones, isassociated with low average transgene expression and high cell-to-cellvariability in transgene expression. The present inventors thereforeasked whether homology probing could be used to identify new selectablemarker proteins that have better performance characteristics than ourexisting selectable marker genes. Specifically, the present inventorsidentified a homolog of the PuroR gene (FIG. 13 ), synthesized a humancodon-optimized version of its cognate open reading frame (PuoR2),cloned it into the expression system used throughout this study, used itto select for a puromycin-resistant cell clones, pooled these clones,and then used flow cytometry to measure the mCherry expression level inthe resulting PuroR2-derived cell line (FIG. 12 ). These experimentsrevealed that PuroR2 is the second-most effective selectable marker geneat driving expression of a linked recombinant protein, generating anaverage mCherry brightness of 16,751, slightly more than that driven inBleoR-selected cells and half that driven by the ER50BleoR marker (FIG.12 ).

Discussion—Part 2

The present inventors hypothesized that each dominant selectable markerestablishes a threshold of transgene expression below which no cell cansurvive, that each marker gene/protein establishes a distinct thresholdof transgene expression, and that this threshold is determined bynumerous properties of the marker protein, some of which are intrinsicto the protein (e.g. binding affinity for the antibiotic, mechanism ofantibiotic inactivation, etc.) while others are extrinsic (e.g. itsability to fold, function and avoid proteolysis in the alien environmentof the mammalian cell). Here the present inventors tested a keyprediction of this hypothesis by creating transgenes in which thedestabilization domains (DDs) from the human estrogen receptor (ER50)and the E. coli DHFR were appended to the N-terminus of the BleoR,PuroR, HygR, BsdR, and NeoR marker proteins, transfecting thesetransgenes into 293F cells, selecting for antibiotic-resistant celllines, then measuring the levels of the linked upstream recombinantprotein (mCherry) in the resulting mixed-clone cell lines. In each case,it was observed that cell lines generated with degron-tagged markerproteins expressed higher and more homogeneous levels of mCherryexpression. Moreover, it was observed that the extent of this effectwas, in general, slightly greater for ecDHFR-tagged markers than forER50-tagged markers.

These experimental results describe significantly improved selectablemarkers for each of the five antibiotics used in mammalian celltransgenesis experiments. BleoR, which was already the best marker forselecting transgenic cell lines with high levels of transgeneexpression, was improved by >2-fold by addition of the ER50 domain,making ER50BleoR the single best marker for selecting cell lines withhigh levels of transgene expression. The next most significantdevelopment was the PuroR2 marker, use of which allows puromycin toselect for cells with the second-highest level of transgene expression,slightly higher than BleoR and ˜2.5-fold higher than PuroR. Even largerincreases of 5- to 6-fold were observed for degron-tagged forms of BsdR,as ER50BsdR- and ecDHFRBsdR-selected cells displayed levels of transgeneexpression similar to that of cells carrying the PuroR or HygR markers,and higher than those selected with any of the three forms of the NeoRmarker. As for the degron-tagged forms of the HygR and NeoR proteins,they also yielded cell lines with higher levels of transgene expression,though the effects were in all cases quite modest (<30% increase). Inshort, this study has generated improved markers for all five majormammalian cell antibiotics, and in particular ER50BleoR for zeocinselection, PuroR2 for puromycin selection, and both ER50BsdR andecDHFRBsdR for blasticidin selection.

DD Stabilizing Drugs Alter the Threshold of Transgene ExpressionEstablished by DD-Tagged SM Genes/Proteins

The present inventors have found that DD-stabilizing drugs alter therelationship between SM function and transgene expression. Morespecifically, the ER50 DD is stabilized by adding 4-hydroxytamoxifen(4-OHT) to the culture media, resulting in a higher number of initialantibiotic-resistant cell clones, with reduced average level oftransgene expression. However, once established, theseantibiotic-resistant cell lines can be passaged in progressively lowerconcentrations of the stabilizing drug, eventually yielding cell linesthat grow well in the absence of 4-OHT. The same is true for cell linesselected with ecDHFR DD-tagged marker, with the exception thattrimethoprim is used instead of 4-OHT. The result is that the DD-taggedmarkers can be used with a sliding scale of stabilizing drug to achievethe desired level of transgene expression.

A Transposon-Based Platform for Engineering Cells and Exosomes

Human cells do not have a natural, endogenous, functional DNAtransposon. However, three different transposons have been developed foruse in human and other mammalian cells: Sleeping Beauty, PiggyBac, andTcBuster. The present inventors chose Sleeping Beauty (SB) transposonsas the platform for transgene delivery. To implement use of SBtransposons, the present inventors synthesized the plasmid pITRSB asshown in FIG. 14 .

The critical features of this plasmid are (i) a gene (e.g.,RSV-SB100x-pAn) designed to express an optimized version of the SBtransposase enzyme, located outside the transposon and (ii) a functionaltransposon comprised of the inverted tandem repeats (ITR-L and ITR-R)that define the left and right ends of the transposon (the transposon iscomprised of all DNA between the 5′most bp of the ITR-L to the 3′most bpof the ITR-R).

This vector works as follows:

-   -   1. One or more transgenes are inserted between the ITR-L and        ITR-R elements using the unique SgrDI and SbfI sites.    -   2. The resulting circular plasmid is transfected into a        mammalian cell line.    -   3. Expression from the RSV LTR drives expression of the SB100X        transposase.    -   4. The SB100X transposase excises the transposon from the        plasmid and inserts it into the host cell genome

The present inventors used this pITRSB vector platform to deliver anexosomal marker-expressing transgene (CMV-CD81-mNeonGreen-2a-PuroR),validating that the system worked as planned. These experiments alsorevealed that transgene delivery via SB transposition led to higherlevels of expression than either plasmid-based delivery orlentivirus-based delivery.

To determine whether transposon-mediated transgene delivery wasrefractory to transgene fragmentation, the present inventors createdderivatives of pITRSB designed to express multiple genes. However, theexpression of SM-expressing genes required some consideration for howbest to express them as separate, stand-alone genes.

Development of Crippled Promoter-Driven SM Genes

The present inventors engineered the SM-expressing gene to be drivenfrom the weak, minimal EF1alpha promoter (EFS), while the reporter genefor monitoring transgene delivery—mCherry—was expressed from the strongCMV promoter. FIG. 15 shows the map of this plasmid designated as“pS179”.

pS179 was transfected into 293F/tet1 cells (to be described below)followed by addition of 3 microgram/ml puromycin. The resultingcolonies, of which there were thousands, were pooled, expanded, andassayed by flow cytometry (FIG. 16 ).

The clear separation of pS179 transfected cell line from the 293F cellline as shown in FIG. 16 represents strong evidence that the vastmajority (>99) of cells in the puromycin-resistant cell line alsoexpressed the mCherry transgene, consistent with the hypothesis that thepuromycin-resistant cell lines generated using this vector are theresult of SB100X-mediated transposition rather than integration offragmented plasmids.

293F Cell Lines for Doxycycline-Inducible Transgene Expression

While it is possible to achieve high-level expression of constitutivelyexpressed transgenes, there are many instances when high-levelconstitutive expression is either undesired or impossible. To overcomethis limitation the present inventors sought to develop Tet-Onderivatives of 293F cells which would allow fortetracycline/doxycycline-inducible expression of transgenes under thecontrol of tet-regulated promoters (e.g. TRE3G).

To pursue this strategy the present inventors combined (A) the existingknowledge of Tet-On gene expression systems, which shows that thertTAv16 protein displays higher Tet-regulated gene expression than anyother Tet-regulated transcription factor, with (B) the information thepresent inventors had learned regarding the effect of selectable marker(SM) on transgene expression as described above, specifically:

-   -   i. to ensure expression of a recombinant protein of interest        (POI) in 100% of antibiotic-resistant cells, it should be        expressed from a CMV-driven, bicistronic ORF that encodes (a)        the POI, followed by (b) a viral 2a peptide and (c) the        selectable marker ORF; and    -   ii. the BleoR and ER50BleoR proteins select for higher-level        expression of a linked, upstream POI than any other markers        (save for PuroR2).

These considerations led the present inventors to create the plasmiddesignated as “pS147” (FIG. 17 ).

Following transfection of cells with this plasmid and selection forzeocin-resistant cells, the rtTAv16 protein will be expressed as a2a-tagged protein, and thus detectable using anti-2a antibodies (the 2aexpression system always appends the first 17 amino acids of the 2apeptide to the C-terminus of the upstream protein). These cell lines aredesignated with the generic term of “Ftet” lines, and these lines arefurther segregated according to whether the cell line was generated bypooling numerous ZeoR clones (designated as “Ftet1”), or by pickingsingle cell clones (SCCs) and screening them for relative expression ofthe rtTAv16-2a protein, with the highest-expressing SCC designated asthe cell line “Ftet2”.

In the course of these experiments it was observed that TRE3G-regulatedtransgenes displayed a pattern of stochastic, doxycycline-independentexpression in Ftet cells, in some cases leading to significant transgeneexpression.

The present inventors created the plasmid designated as “pCG210”, whichis identical to pS147 except that it encodes rtTAv16/G72P with a singleamino acid substitution. pCG210 was transfected into 293F cells,selected in zeocin-containing media, followed by (a) pooling ofthousands of cell clones to make the Ftet3 cell line, and (b) cloning ofthe 10 fastest-growing single cell clones (SCCs).

In addition, the present inventors combined the ER50BleoR marker withboth rtTAv16/G72 and rtTAv16 to create two additional vectors(designated as “pCG211” and “pCG212” designed to drive even higher-levelexpression these transcription factors. These plasmids were transfectedinto 293F cells followed by selection in zeocin-containing mediasupplemented with 4-hydroxytamoxifen to stabilize the ER50 degron. Onceagain, pooled cell lines and single cell clones were derived from eachof these transfections.

To assess the levels and homogeneity of rtTAv16-2a or rtTAv16/G72P-2a inthese various cell lines, the present inventors processed them forimmunofluorescence microscopy using antibodies specific for the viral 2apeptide epitope tag. These cell lines varied significantly in theirlevels of linked protein expression (FIG. 18 ). Moreover, theseobservations provide important insights into the present system fordriving high-level expression of recombinant proteins:

-   -   i. In all three pooled cell lines, the levels of anti-2a        staining appeared well above background in the vast majority of        cells    -   ii. Staining appeared, on average, to be brighter for the pools        generated using the ER50BleoR marker, even though at this stage        of analysis the cells had been continuously supplemented with        4-hydroxy-tamoxifen (4OHT)—levels should rise significantly once        the 4OHT is removed by selecting for the clones with highest        original expression and/or amplification of the introduced        transgene.    -   iii. However, the levels of expression in the SCCs showed that        the CG210 SCCs expressed extremely high levels of 2a-reactive        protein, on average seemingly higher than the CG211 SCCs.    -   iv. Among the CG211 SCCs, clones 3 and 4 appeared brightest,    -   v. Among the CG210 SCCs, clone 7 appeared brightest, and likely        brightest of all, even the CG211 clones. In addition, it had the        least aberrant morphology of all of these clones.    -   vi. There appeared to be high variation in staining within each        clone, with notably higher staining in areas of the coverglass        where cell density was lowest, which might be explained by:        -   a. Reduced competition for antibody at the periphery of the            coverglasses;        -   b. Reduced expression from the CMV enhancer when cells were            at high density; or        -   c. Actual clonal heterogeneity

Vectors for Inducible Transgene Expression

Based on the above developments, the present inventors created andvalidated a vector platform that combines: (a) engineered selectablemarker genes that drive high-level recombinant protein expression; (b)Sleeping Beauty transposition, which delivers transgenes at highefficiency; and (c) a doxycycline-inducible transgenes, using a promoter(TRE3G) that can drive high-level, rtTA-mediated, dox-inducibletransgene expression.

These features are evident in the plasmid designated as “pS180” (FIG. 19). This plasmid is designed to mobilize a SB transposon that integratesinto the host cell chromosome, carrying: (a) the EFS-PuroR transgene forselection of transgene-expressing cells and (b) a TRE3G-mCherrytransgene for measurement of basal and doxycycline-induced transgeneexpression.

To validate its utility for driving high-level, doxycycline-inducibletransgene expression, the present inventors transfected p5180 into Ftet1cells, selected for puromycin-resistance, pooled all colonies to form asingle, mixed-clone cell line, and then assayed mCherry expression byflow cytometry after 1 day of culture in normal media or mediasupplemented with 100 ng/ml doxycycline (FIG. 20 ). These data indicatethat the addition of dox induced mCherry expression by ˜50-fold,validating the present approach.

In the course of these experiments, the present inventors asked whetherthe CMV enhancer/promoter might also be inducible, not by doxycyclinebut by existing signaling pathways that can be activated in 293F cells.In particular, it was found that the CMV enhancer has a number ofbinding sites for the transcription factors NFAT and NF-kB, a pair oftranscription factors that are known to be activated by protein kinase C(PKC). Furthermore, the natural product prostratin is a potent activatorof conventional PKC isoforms. To assess its ability to drive higherlevels of transgene expression from the CMV promoter, the presentinventors assayed mCherry expression by flow cytometry in Ftet1 cellsgrown in the presence or absence of prostratin (FIG. 21 ).

These results indicate that addition of prostratin elevates mCherryexpression nearly 2-fold. By deduction, these results indicate thataddition of prostratin might also elevate expression of the rtTAv16protein in Ftet cells, as the expression of the rtTAv16 protein in thesecell lines is under the control of the CMV promoter. Furthermore, sincethe Tet-regulated mCherry gene in Ftet1/pS180 cells shows detectablelevels of dox-independent expression (FIG. 22 ), addition of prostratinwould be expected to induce mCherry expression from the TRE3G promoterin Ftet cells carrying the pS180-mobilized transposon. Here the presentinventors tested this prediction by incubating the Ftet1/pS180 cell linein the presence of prostratin, doxycycline, or prostrating+doxycycline,and then measuring mCherry expression by flow cytometry (FIG. 22 ).

The resulting data establish that addition of prostratin alone elevatesmCherry expression ˜2-fold, and that addition of prostratin furtherincreases doxycycline-induced gene expression by ˜15%.

Marker-Driven Expression of Transposon-Delivered Transgenes

The present inventors next tested whether the choice of selectablemarker affected the levels of transgene expression from Sleeping Beautytransposons. Using the TRE3G-mCherry transgene as reporter, the PuroRcoding region in the EFS-PuroR gene was replaced with the ER50PuroR andecDHFRPuroR markers, the resulting plasmids were transfected into Ftet1cells, and then the cells were placed in puromycin-containing media, insome cases supplemented with appropriate concentrations of4-hydroxy-tamoxifen (4OHT) or trimpethoprim, drugs that stabilize theER50 and ecDHFR destabilization domains, respectively (transfection withthe plasmid carrying the EFS-ecDHFRPuroR marker gene yielded nopuromycin-resistant colonies either in the presence or absence oftrimethoprim).

Transfection with the plasmid carrying the EFS-ER50PuroR marker yieldedcolonies in the presence of 4OHT but not in its absence. Theseselections were carried out at 1 microgram/ml puromycin. Cellstransfected with the PuroR-based plasmid were selected at both 1 and 3microgram/ml puromycin. The three cell lines arising from thesetransfections were then cultured in the presence of 100 ng/mldoxycycline for 24 hours and assayed by flow cytometry (FIG. 23 ). Theseexperiments revealed that the basal, leaky expression of mCherry fromthe TRE3G promoter was lowest for cells selected with the PuroR marker(plasmids pS180) at 1 ug/ml puromycin (relative brightness of 150),slightly higher when the same set of transfected cells had been selectedat 3 ug/ml puromycin (relative brightness of 199), but highest for cellsselected using the ER50PuroR marker (plasmid pS240) and 1 ug/mlpuromycin (relative brightness of 593), which was ˜4-fold higher thanobserved for the PuroR-selected cell line.

These data support the present inventors' operating hypothesis ofselectable marker function. Moreover, these data show that elevatingantibiotic concentration elevates the threshold of transgene expressionneeded for antibiotic resistance, though only slightly. It should alsobe noted that these mCherry expression levels reflect leaky expressionfrom the TRE3G promoter in the absence of doxycycline and are thereforelikely reporting on differences in gene dosage that are selected by eachcombination of selectable marker and antibiotic concentration.

To explore these conclusions further, the present inventors created aseries of vectors using the NeoR and BsdR markers and theirdegron-tagged derivatives. In brief, these experiments revealed, onceagain, the effect of degron tagging on transgene expression, as293F/Ftet2 cells selected with the NeoR marker showed ˜1.5-fold highermCherry expression when grown in a 2x concentration of G418 (800 μg/ml),and Ftet2 cells selected with the ER50NeoR gene in the absence of 4OHTshowed mCherry expression levels twice again as high (FIG. 24 ). Notsurprisingly, Ftet2 cells selected using the BsdR marker showed thelowest level of mCherry expression, though this rose in cells selectedwith the ER50BsdR marker (FIG. 25 ).

The above results support the hypothesis that cell and exosomeengineering can be best achieved using Sleeping Beauty-based genedelivery and doxycycline-induced expression of recombinant proteins ofinterest.

Vectors and Cell Lines for Inducible Expression of SARS-CoV-2 Spike (S),Nucelocapsid (N), Membrane (M), and Envelope (E) Proteins, and VLPs.

Based on the above information, the present inventors generated a singleSleeping Beauty transposon carrying 5 genes, one that expresses thePuroR protein from a crippled EF1alpha promoter, and 4 other genes inwhich codon-optimized versions of the S, N, M, and E ORFs are drivenfrom the dox-inducible TRE3G promoter (FIG. 25 ).

This plasmid, designated as “pS226”, also carries 4 amino acidsubstitution mutations that are designed to prevent furin-mediatedcleavage at the S1/S2 junction site, and two additional amino acidsubstitution mutations that are believed to stabilize the trimeric formof the Spike protein.

The plasmid pS226 was transfected into Ftet2 cells, a single cell clonethat was generated by transfection of 293F cells with the plasmid pS147and which grows in media containing 200 ug/ml zeocin. Two days later,half the cells were placed in media containing 1 μg/ml puromocyin andhalf were placed in media containing 3 μg/ml puromycin. No coloniesarose on the plates fed with the higher concentration of puromycin, butmany clones arose on cells fed with media containing 1 μg/ml puromycin.

These puromycin-resistant clones were pooled and expanded leading to thefirst master cell bank. In test experiments, addition of doxycycline tothe culture media led to expression of all 4 proteins, as well as theappearance of all 4 proteins in exosome/VLP fraction generated by eitherof two purification protocols (differential centrifugation andfiltration/chromatography, respectively).

For VLP production, cells carrying the S/N/M/E-expressing transposonsare resuspended in freestyle media supplemented with 800 ng/mldoxycycline, prostratin, and forskolin at a density of0.5-1×10{circumflex over ( )}6 cells/ml and cultured with shaking for 3days.

For VLP purification, the culture media was spun at 500×g for 5 minutes,the SN transferred to a fresh tube, spun again at 5000×g for 10 minutes,followed by gravity-flow filtration of the SN through a 0.22 micron poresize PES filter sterilization unit (125, 250, or 500 ml capacity forlarge surface area filtration). The filtrate, which is referred to as aclarified tissue culture supernatant (CTCS) was then concentrated usinga Centricon 70 spin concentrator with a 100 kDa pore size cutoff. Thus,starting CTCS of 70, 140, or 210 mls can be reduced to a volume of 0.5ml. This material is then loaded on qEV size exclusion chromatographycolumn (Izon) and 0.5 mls fractions are collected. Fractions 1-3 are thevoid volume, fractions 4-6 contain the VLPs, and subsequent fractionscontain proteinaceous contaminants. The resulting 1.5 mls are pooled andassayed for protein concentration and numbers of and sizes of vesiclesin the sample.

The resulting VLP preps can be used for biological studies of virus-hostinteraction, but also as the key material of a vaccine.

The present inventors also generated a plasmid designated “pS225”, whichis identical to pS226, except for the fact that pS226 expresses theWuhan-1 strain Spike protein, and a plasmid designated “pCG201”, whichis identical to pS226, except for the fact that pCG201 expresses theD614G Spike protein (only 1 amino acid difference from the Wuhan-1strain Spike protein). SARS-CoV-2 VLPs were made by transfection of the293F/pS147 SCC with pS225, pS226, or pCG201, each of which are designedfor transposon-mediated delivery of dox-regulated genes expressing theSARS-CoV-2 S, N, M, and E proteins.

F/Tet-on Cell Lines that Make SARS-CoV-2 VLPs: Expression Data(Immunoblot)

The two cell lines (293F/pS147/pS225 and 293F/pS147/pS226) weresuspended in Freestyle media in the presence of 100 ng/ml dox, incubatedin TC shaker flasks for 3 days, with aliquots removed at d0 and d2. Celllysates=0d, 2d, and 3d lanes. Exosomes/VLPs were collected from the TCSN at d3. Immunoblots (FIG. 27 ) are with rabbit polyclonal seraspecific for C-terminal peptides of the S, M, and E proteins, and arabbit monoclonal specific for the N protein.

F/Tet-on Cell Lines that Make SARS-CoV-2 VLPs: Ultrastructural Analysis

FIG. 28 shows the negative stain electron microscopy of VLPspreparations purified from the TC SN of 293F/pS147/pS226 cell culturesafter three days incubation in Freestyle media supplemented withdoxycycline. Note the distinctive morphology of Spike protein trimersextending from the VLP surface.

Mice and Immunization Schedule

TABLE 7 Concentration Type of cell donor 4 × 10{circumflex over ( )}8/ul293F ps226 derived VLP (10 uL) Group 1 VLP dose 7.5 ug 6 Group 2 VLPdose 1.75 ug 6 Group 3 VLP dose 0.35 ug 6 Group 4 Exosomes 4 Group 5Control 4 Total mice 26

TABLE 8 Exo Conc (particle/ml) VLP Conc (particle/ml) High dose 9 × 10e84 × 10e8 Injected/mice 1.80E+07 1.72E+07

The disclosure of every patent, patent application, and publicationcited herein is hereby incorporated herein by reference in its entirety.The citation of any reference herein should not be construed as anadmission that such reference is available as “prior art” to the instantapplication. Throughout the specification the aim has been to describethe preferred embodiments of the invention without limiting theinvention to any one embodiment or specific collection of features.Those of skill in the art will therefore appreciate that, in light ofthe instant disclosure, various modifications and changes can be made inthe particular embodiments exemplified without departing from the scopeof the present invention. All such modifications and changes areintended to be included within the scope of the appended claims.

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1. A non-naturally occurring selectable marker (SM) protein, wherein theSM protein comprises a destabilization domain (DD) operably connected toa SM protein, thereby providing a non-naturally occurring SM protein. 2.The non-naturally occurring SM protein according to claim 1, whereinsaid SM protein is an SM protein that functions in a mammalian cell. 3.The non-naturally occurring SM protein according to claim 1, whereinsaid DD is appended to the N-terminus of said SM protein, the C-terminusof said SM protein, or both the N-terminus and the C-terminus of said SMprotein.
 4. The non-naturally occurring SM protein according to claim 1,wherein said DD is appended to the N-terminus of said SM protein.
 5. Thenon-naturally occurring SM protein according to claim 1, wherein said SMprotein is a dominant SM protein.
 6. The non-naturally occurring SMprotein according to claim 1, wherein said non-naturally occurring SMprotein confers resistance to zeocin, puromycin, hygromycin, G418,and/or blasticidin.
 7. The non-naturally occurring SM protein accordingto claim 6, wherein said SM protein that confers resistance to zeocin isBleoR, wherein said SM protein that confers resistance to puromycin isPuroR; wherein said SM protein that confers resistance to hygromycin isHygR; wherein said SM protein that confers resistance to G418 is NeoR;and/or wherein said SM protein for mammalian cells that confersresistance to blasticidin is BsdR.
 8. The non-naturally occurring SMprotein according to claim 1, wherein said DD is derived from the humanestrogen receptor (ER50), thereby providing a SM protein operablyconnected to the ER50(DD).
 9. The non-naturally occurring SM proteinaccording to claim 8, wherein said SM protein operably connected to theER50(DD) is BleoR operably connected to the ER50(DD), i.e., ER50BleoR;PuroR operatively connected to the ER50(DD), i.e., ER50PuroR; HygRoperatively connected to the ER50(DD), i.e., ER50HygR; NeoR operativelyconnected to the ER50(DD), i.e., ER50NeoR; or BsdR operatively connectedto the ER50(DD), i.e., ER50BsdR.
 10. The non-naturally occurring SMprotein according to claim 1, wherein said DD is derived from theEscherichia coli dihydrofolate reductase (ecDHFR), thereby providing anSM protein operatively connected to the ecDHFR(DD).
 11. Thenon-naturally occurring SM protein according to claim 10, wherein saidSM operatively linked to the ecDHFR(DD) is BleoR operatively linked tothe ecDHFR(DD), i.e., ecDHFRBleoR; PuroR operatively linked to theecDHFR(DD), i.e., ecDHFRPuroR; HygR operatively linked to theecDHFR(DD), i.e., ecDHFRHygR; NeoR operatively linked to the ecDHFR(DD),i.e., ecDHFRNeoR; or BsdR operatively linked to the ecDHFR(DD), i.e.,ecDHFRBsdR.
 12. The non-naturally occurring SM protein according toclaim 1, wherein the engineered SM protein further comprises an alteredamino acid sequence resulting from a frameshift mutation within anucleotide sequence that encodes the last about 10, 20, 30, 40, or 50amino acids at the 3′ end of the DD-tagged SM.
 13. An isolated nucleicacid, the nucleotide sequence of which encodes the engineered SM proteinaccording to claim
 1. 14. An expression vector comprising a nucleicacid, the nucleotide sequence of which encodes a selectable marker (SM)protein, optionally an unstable and/or degraded SM protein, and anoperably linked recombinant protein of interest (POI), wherein thenucleic acid is operably linked to an expression control sequence.15-16. (canceled)
 17. The expression vector according to claim 14,wherein said nucleic acid comprises an open reading frame (ORF) thatencodes (a) the POI, followed by (b) a self-cleaving peptide which caninduce ribosomal skipping during translation, and (c) the SM protein.18-28. (canceled)
 29. A cell comprising the expression vector of claim14.
 30. A cultured cell line comprising the expression vector of claim14, wherein the cells in the cultured cell line are selected byculturing in a selection-containing media. 31-35. (canceled)
 36. Amethod of making extracellular vesicles (EVs) comprising culturing acell line of claim 30, wherein the cell line produces EVs comprising oneor more of the POIs, and isolating the EVs produced.
 37. The methodaccording to claim 36, wherein said EVs are exosomes or microvesicles.38-43. (canceled)
 44. An expression vector comprising: (a) a nucleicacid the nucleotide sequence of which encodes a transposon comprised ofinverted terminal repeats (ITR-L and ITR-R elements) that define theleft and right ends of the transposon; (b) one or more genes of interest(GOIs) encoding one or more proteins of interest (POIs) inserted betweenthe ITR-L and ITR-R elements, wherein the GOIs are operably linked toexpression control sequences; and (c) a nucleic acid the nucleotidesequence of which encodes a transposase enzyme, wherein the nucleic acidis located outside the transposon, and wherein the nucleic acid isoperably linked to an expression control sequence. 45-133. (canceled)